Tetrol steroids and esters

ABSTRACT

The invention provides compositions comprising formula 1 steroids, e.g., 16α-bromo-3β-hydroxy-5α-androstan-17-one hemihydrate and one or more excipients, typically wherein the composition comprises less than about 3% water. The compositions are useful to make improved pharmaceutical formulations. The invention also provides methods of intermittent dosing of steroid compounds such as analogs of 16α-bromo-3β-hydroxy-5α-androstan-17-one and compositions useful in such dosing regimens. The invention further provides compositions and methods to inhibit pathogen (viral) replication, ameliorate symptoms associated with immune dysregulation and to modulate immune responses in a subject using certain steroids and steroid analogs. The invention also provides methods to make and use these immunomodulatory compositions and formulations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 37 C.F.R. 1.53(b)and claims priority of U.S. application Ser. No. 09/535,675, filed Mar.23, 2000, now U.S. Pat. No. 6,667,299 B1, which is acontinuation-in-part of abandoned U.S. application Ser. No. 09/414,905,filed Oct. 8, 1999 and this application claims priority from abandonedU.S. provisional application Ser. No. 60/190,140, filed Mar. 16, 2000,abandoned U.S. provisional application Ser. No. 60/164,048, filed Nov.8, 1999, abandoned U.S. provisional application Ser. No. 60/140,028,filed Jun. 16, 1999, and abandoned U.S. provisional application Ser. No.60/126,056, filed Mar. 23, 1999, all of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

The invention relates to methods to make and use steroids, such as16α-bromo-3β-hydroxy-5α-androstane-17-one (16α-bromoepiandrosterone orhereafter “BrEA”) and new analogs thereof. The steroids are useful for anumber of therapeutic and non-therapeutic applications, including theiruse as immune modulators. The present invention also relates to methodsto make the compounds, compositions and formulations.

BrEA and its preparation from the steroid compound3β-hydroxyandrost-5-en-17-one (dehydroepiandrosterone or “DHEA”) havebeen described (see, e.g., J. Org. Chem. 1962 27:2937-2938). Methods toprepare DHEA and other steroids and their biological properties havebeen described, see, e.g., U.S. Pat. Nos. 2,833,793, 2,911,418,3,148,198, 3,471,480, 3,976,691, 4,268,441, 4,427,649, 4,542,129,4,666,898, 4,956,355, 5,001,119, 5,043,165, 5,077,284, 5,028,631,5,110,810, 5,157,031, 5,162,198, 5,175,154, 5,277,907, 5,292,730,5,296,481, 5,372,996, 5,387,583, 5,407,684, 5,424,463, 5,461,042,5,478,566, 5,506,223, 5,518,725, 5,527,788, 5,527,789, 5,532,230,5,559,107, 5,562,910, 5,583,126, 5,585,371, 5,587,369, 5,591,736,5,593,981, 5,610,150, 5,635,496, 5,641,766, 5,641,768, 5,656,621,5,660,835, 5,686,438, 5,696,106, 5,700,793, 5,707,983, 5,709,878,5,710,143, 5,714,481, 5,728,688, 5,736,537, 5,744,462, 5,753,237,5,756,482, 5,776,921, 5,776,923, 5,780,460, 5,795,880, 5,798,347,5,798,348, 5,804,576, 5,807,848, 5,807,849, 5,811,418, 5,824,313,5,824,668, 5,824,671, 5,827,841, 5,837,269, 5,837,700, 5,843,932,5,846,963, 5,859,000, 5,872,114 and 5,872,147; German patent numbers2035738 and 2705917; PCT publication numbers WO 95/21617, WO 97/48367,WO 98/05338, WO 98/50040, WO 98/50041, WO 98/58650; European publicationnumber 0020029; Ben-David, et al., Proc. Soc. Expt. Biol. Med. 1967125:1136-1140, Coleman et al., Diabetes 1982 31:830, Oertel, et al., J.Steroid Biochem. 1972 3:493-496, Pashko, et al., Carcinogenesis 19812:717-721, Schwartz et al., Nutr. Cancer 1981 3:46-53; Dyner et al., J.Acquired Immune Deficiency Syndromes 1993 6:459-465; A. A. Afanasii andY. A. Titov, Total Steroid Synthesis, Plenum Press, New York, 1970, see,e.g., p 1-304.

The use DHEA and other steroids in various applications, e.g.,modulating immune responses have been described, e.g., U.S. Pat. Nos.5,869,090, 5,863,910, 5,856,340, 5,824,668, 5,804,576, 5,753,237,5,714,481, 5,709,878, 5,407,684, 5,206,008, 5,077,284, 4,978,532,4,898,694, 4,542,129, 3,711,606 and 3,710,795. U.S. Pat. No. 4,956,355and PCT publication number WO 97/48367, have described the use of BrEAand certain steroid compounds to treat certain virus or bacterialinfections, such as human immunodeficiency virus (“HIV”) infection.

Various biological effects and/or metabolic conversions of steroidcompounds have been described, e.g., Batta et al., J. Biol. Chem. 198625:127-133, Belli et al., Liver 1991 11:162-169, Bhattacharjee et al.,Anal. Biochem. 1992 201:233-236, Blake et al., Int J. Peptide ProteinRes. 1982 20:97-101, 1986 25:127-133, Bonaventura, Am. J. Obstet.Gynecol. 1978 131:403-409, Bucala et al., J. Steroid Biochem. 198625:127-133, Carey et al., Biochem. 1981 20:3637-3648, Chen et al.,Carcinogenesis 1999 20:249-254, Chen et al., Carcinogenesis 199819:2187-2193, Chow et al., Antisense Res. Dev. 1994 4:81-86, Citro etal., Dis. Colon Rectum 1994 37(2 Suppl):S127-S132, Cleary, Proc. Soc.Exp. Biol. Med. 1991 196:8-16, Cleary, Int. J. Biochem. 1990 22:205-210,Crawford et al., Lab. Invest. 1994 71:42-51, Danenberg et al.,Antimicrob. Agents Chemother. 1992 36:2275-2279, Dotzlaw et al., CancerRes. 1999 59:529-532, Falany et al., J. Steroid Biochem. Mol. Biol. 199448:369-375, Faredin et al., J. Investigative Dermatol. 1969 52:357-361,Galigniana et al., Mol. Pharmacol. 1999 55:317-323, Goto et al., J.Chromatogr. 1983 276:289-300, Grenot Biochem. 1992 31:7609-7621,Hofbauer et al., Life Sci. 1999 64:671-679, Huijghebaert et al., J.Lipid Res. 1986 27:742-752, Hurd et al., Oncogene 1999 18:1067-1072,Iida et al., J. Lipid Res. 1995 36:628-638, Jellinck et al., Steroids1967 10:329-346, Jonsson et al., J. Pediatr. Gastroenterol. Nutr. 199520:394-402, Kalimi et al, Mol. Cell. Biochem. 1994 131:99-108, Kramer etal., J. Biol. Chem. 1994 269:10621-10627, LaRochelle et al., Steroids1984 43:209-217, Liao et al., Carcinogenesis 1998 19:2173-2180,Lillienau et al., J. Clin. Invest. 1992 89:420-431, Loria,Psychoneuroendocrinology 1997 22:S103-S108, Luscher et al Mol. Immunol.1983 20:1099-1105, Manna et al., J. Biol. Chem. 1999 274:5909-5918,Marschall et al., J. Biol. Chem. 1989 264:12989-12993, Medh et al.,Cancer Res. 1998 15:3684-3693, Mohan et al., Steroids 1992 57:244-247,Munoz de Toro et al., J. Steroid Biochem. Mol. Biol. 1998 67:333-339,Padgett et al., J. Neuroimmunol. 1998 84:61, Padgett et al., Ann. N.Y.Acad. Sci. 1995 774:323, Padgett et al., J. Immunol. 1994 153:1544-1552,Pashko et al., Carcinogenesis 1984 5:463-466, Pashko et al.,Carcinogenesis 1981 2:717, Petrylak et al., J. Clin. Oncology 199917:958-967, Podesta et al., Steroids 1996 61:622-626, Regelson et al.,Ann. N.Y. Acad. Sci. 1994 719:564, Schmassmann et al., Gastroenterology1993 104:1171-1181, Schmassmann et al., Hepatology 1990 11:989-996,Schreiber et al., Lancet 353:459-461, Schreiber, Neth. J. Med. 199853:S24-31, Schwartz et al., Cancer Res. 1988 48:4817, Shahidi et al.,Biochem. Biophys. Res. Commun. 1999 254:559-565, Steer et al., Ann.Rheum. Dis. 1998 57:732-737, Suzuki et al., Steroids 1998 63:672-677,Suzuki et al., Steroids 1996 61:296-301, Swaan et al., BioconjugateChem. 1997 8:520-525, Tang et al, Anticancer Drug Res. 1998 13:815-824,Thomas et al., J. Steroid Biochem. 1986 25:103-108, Utsumi et al.,Cancer Res. 1999 59:377-381, Vanden Heuvel, J. Nutr. 1999 129(2SSuppl.):575S-580S, Wang et al., Endocrinology 1998 139:3903-3912, Wonget al., J. Biol. Chem. 1999 274:5443-5453, Xie et al., Endocrinology1999 140:219-227, Yen et al., Lipids 1977 12:409-413, Zackheim et al.,Arch. Dermatology 1998 134:949-954, Zhang et al., Biochim. Biophys. Acta1991 1096:179-186, Zhu et al., Carcinogenesis 1988 19:2101-2106.

Compositions containing BrEA that were used to deliver the compound tocells or cell extracts usually included a significant amount of water.Such compositions contained solvents such as dioxane ordimethylsulfoxide (“DMSO”), which contained water, or an aqueouscyclodextrin solutions to facilitate compound delivery to cells, see,e.g., J. Pharmacol Exp. Ther. 1998, 285:876-83, Cancer Res. 198646:3389-95, Carcinogenesis 1985 6:333-35, Carcinogenesis 1981 2:717-721,Carcinogenesis 1981 2:683-86. Such compositions are typically deliveredto animals by injection or to cells in tissue culture by addition tocell culture medium. European publication number EP 429 187 describesformulations that contain DHEA or BrEA and polyvinylpyrrolidone andcrosslinked polyvinylpyrrolidone. Some of these compositions may haveundesired or suboptimal properties. For example, solvents such asdioxane, DMSO or chloroform are generally not preferred or suitableparenteral excipients, particularly for human use. Formulations thatcontain BrEA or related steroids and that have improved properties,e.g., lower toxicity, improved chemical stability or desirablecharacteristics for large-scale synthesis are needed.

Mammalian immune responses to infections or other conditions are oftencharacterized by responses mediated by different effector cellpopulations. In some situations, helper T cells designated Th1 in themurine system, facilitate immune effector functions that are typicallydominated by cell-mediated responses. In other cases, helper T cellsdesignated Th2 cells facilitate immune effector functions that aretypically dominated by humoral responses. A vigorous Th1 response isusually needed to clear infections or to slow the progression of aninfection. When a subject's immune response is biased to, or dominatedby, a Th2-type response, the cytokines associated with the Th2 responsetend to suppress the immune system's capacity to mount a vigorous Th1response at the same time. The converse is also generally true. Whenmammalian immune responses begin to result in an increasing Th2response, the Th1 response to the same condition tends to weaken. WeakTh1 responses may be associated with progression of some infections orother conditions, see, e.g., M. Clerici and G. M. Shearer, Immunol.Today 14:107-111, 1993; M. Clerici and G. M. Shearer, Immunol. Today15:575-581, 1994. The invention provides compounds and compositionsuseful to enhance Th1 immune responses.

OBJECTS OF THE INVENTION

The invention compositions, formulations or methods accomplish one ormore of the following objects.

One object of the invention is to provide new steroid compounds oranalogs that are suitable for therapeutic and other applications, suchas immune modulators. Invention objects further include providing BrEAhemihydrate (BrEA₂.H₂O), compositions that comprise BrEA hemihydrate andmethods to make and use it. Another object of the invention is toprovide liquid compositions and formulations that comprise a formula 1compound(s), and that comprise about 3% (v/v) or less of water. Anotherobject is to provide compositions one can use as intermediates toprepare human pharmaceutical and veterinary formulations containing aformula 1 compound(s). Another object is to provide intermittent dosingmethods to deliver a formula 1 compound to a subject to enhance Th1immune responses. Further objects are to provide methods to modulateinnate immunity or to enhance Th1 immune responses in a subject byadministering to the subject a formula 1 compound(s) such as BrEA. Otherobjects are to provide methods to inhibit pathogen, e.g., viral,replication in a subject by administering to the subject a formula 1compound(s) such as BrEA. Invention objects include providing formula 1compounds or formulations useful to ameliorate one or more symptoms of apathological condition associated with immune suppression or withdeficient Th1 immune responses. Other objects are to provide methods tomake and use compositions and formulations comprising a formula 1compound(s).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an FTIR (Fourier transform infrared) spectrum obtained by USPmethod <197> of BrEA hemihydrate that was prepared by precipitation ofBrEA from ethanol and water.

FIG. 2 is a FTIR spectrum obtained by USP method <197> of anhydrous BrEAthat was prepared by precipitation of BrEA from anhydrous methanol.

FIG. 3 shows a DSC endotherm of BrEA hemihydrate that was prepared byprecipitation of BrEA from ethanol and water.

FIG. 4 shows a DSC endotherm of anhydrous BrEA that was prepared byprecipitation of BrEA from anhydrous methanol.

FIG. 5 is an XRD (powder X-ray diffraction) spectrum of BrEA hemihydratethat was prepared by precipitation of BrEA from ethanol and water.

FIG. 6 is a FTIR spectrum obtained by USP method <197> of BrEAhemihydrate that was prepared by precipitation of BrEA from acetone andwater.

SUMMARY OF THE INVENTION

In accordance with the objects, the invention provides BrEA hemihydrate

which is optionally characterized by reference to one or more physicalproperties such as its melting point, infrared absorption spectrum orits powder X-ray diffraction spectrum.

Related embodiments include BrEA hemihydrate and one or more excipientssuitable for human pharmaceutical use or for veterinary use. Anotherrelated embodiment is a method to make BrEA hemihydrate comprisingprecipitating BrEA from a solution comprising ethanol and water.

Invention embodiments include a composition comprising a compound offormula 1

and one or more nonaqueous liquid excipients, wherein the compositioncomprises less than about 3% v/v water and wherein,

R¹, R², R³, R⁴, R⁵, R⁶ and R¹⁰ independently are —H, —OR^(PR), —SR^(PR),N(R^(PR))₂, —O—Si—(R¹³)₃, —CN, —NO₂, an ester, a thioester, aphosphoester, a phosphothioester, a phosphonoester, a phosphiniester, asulfite ester, a sulfate ester, an amide, an amino acid, a peptide, anether, a thioether, an acyl group, a thioacyl group, a carbonate, acarbamate, a thioacetal, a halogen, an optionally substituted alkylgroup, an optionally substituted alkenyl group, an optionallysubstituted alkynyl group, an optionally substituted aryl moiety, anoptionally substituted heteroaryl moiety, an optionally substitutedmonosaccharide, an optionally substituted oligosaccharide, a nucleoside,a nucleotide, an oligonucleotide, a polymer, or,

one more of R¹, R², R³, R⁴, R⁵, R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸ are ═O or ═Sand the hydrogen atom that is bonded to the same carbon atom is absent,or,

R³ and both R⁴ together comprise a structure of formula 2

R⁷ is —CHR¹⁰—, —CHR¹⁰—CHR¹⁰—, —CHR¹⁰—CHR¹⁰—CHR¹⁰—, —CHR¹⁰—O—CHR¹⁰—,—CHR¹⁰—S—CHR¹⁰—, —CHR¹⁰—NR^(PR)—CHR¹⁰—, —O—, —O—CHR¹⁰—,—S—NR^(PR)—CHR¹⁰—;

R⁸ and R⁹ independently are —CHR¹⁰—, —CHR¹⁰—CHR¹⁰—, —O—, —O—CHR¹⁰—, —S—,—S—CHR¹⁰—, —NR^(PR)— or —NR^(PR)—CHR¹⁰—, or R⁸ or R⁹ independently isabsent, leaving 5-membered ring;

R¹³ independently is C₁₋₆ alkyl;

R¹⁶ independently are —CH₂—, —O—, —S— or —NH—;

D is a heterocycle or a 4-, 5-, 6- or 7-membered ring that comprisessaturated carbon atoms, wherein 1, 2 or 3 ring carbon atoms of the 4-,5-, 6- or 7-membered ring are optionally independently substituted with—O—, —S— or —NR^(PR)— or where 1, 2 or 3 hydrogen atoms of theheterocycle or where 1 or 2 hydrogen atoms of the 4-, 5-, 6- or7-membered ring are substituted with —OR^(PR), —SR^(PR), —N(R^(PR))₂,—O—Si—(R¹³)₃, —CN, —NO₂, an ester, a thioester, a phosphoester, aphosphothioester, a phosphonoester, a phosphiniester, a sulfite ester, asulfate ester, an amide, an amino acid, a peptide, an ether, athioether, an acyl group, a thioacyl group, a carbonate, a carbamate, athioacetal, a halogen, an optionally substituted alkyl group, anoptionally substituted alkenyl group, an optionally substituted alkynylgroup, an optionally substituted aryl moiety, an optionally substitutedheteroaryl moiety, an optionally substituted monosaccharide, anoptionally substituted oligosaccharide, a nucleoside, a nucleotide, anoligonucleotide or a polymer, or,

one more of the ring carbons are substituted with ═O or ═S,

or D comprises two 5- or 6-membered rings, wherein the rings are fusedor are linked by 1 or 2 bonds.

In other embodiments, the invention provides a compound of formula 1,wherein two or three of R⁷, R⁸ and R⁹ independently are not —CHR¹⁰—,wherein the compound is optionally present in a composition thatcomprises one or more excipients suitable for human pharmaceutical useor for veterinary use.

Invention embodiments also include a compound of formula 1

wherein,

-   -   R¹, R², R³, R⁴, R⁵, R⁶ and R¹⁰ independently are —H, —OR^(PR),        —SR^(PR), —N(R^(PR))₂, —O—Si—(R¹³)₃, —CN, —NO₂, an ester, a        thioester, a phosphoester, a phosphothioester, a phosphonoester,        a phosphiniester, a sulfite ester, a sulfate ester, an amide, an        amino acid, a peptide, an ether, a thioether, an acyl group, a        thioacyl group, a carbonate, a carbamate, a thioacetal, a        halogen, an optionally substituted alkyl group, an optionally        substituted alkenyl group, an optionally substituted alkynyl        group, an optionally substituted aryl moiety, an optionally        substituted heteroaryl moiety, an optionally substituted        monosaccharide, an optionally substituted oligosaccharide, a        nucleoside, a nucleotide, an oligonucleotide, a polymer, or,

one, two or more of R¹, R², R³, R⁴, R⁵, R⁶ and R¹⁰ independently are ═Oor ═S and the hydrogen atom that is bonded to the same carbon atom isabsent, or,

R³ and R⁴ together comprise a structure of formula 2

R⁷ is —CHR¹⁰—, —CHR¹⁰—CHR¹⁰—, —CHR¹⁰—CHR¹⁰—CHR¹⁰—, —CHR¹⁰—O—CHR¹⁰—,—CHR¹⁰—S—CHR¹⁰—, —CHR¹⁰—NR^(PR)—CHR¹⁰—, —O—, —O—CHR¹⁰—, —S—, —S—CHR¹⁰—,—NR^(PR)— or —NR^(PR)—CHR¹⁰—;

R⁸ and R⁹ independently are —CHR¹⁰—, —CHR¹⁰—CHR¹⁰—, —O—, —O—CHR¹⁰—, —S—,—S—CHR¹⁰—, —NR^(PR)— or —NR^(PR)—CHR¹⁰—, or R⁸ or R⁹ independently isabsent, leaving a 5-membered ring;

R¹³ independently is C₁₋₆ alkyl;

D is a heterocycle or a 4-, 5-, 6- or 7-membered ring that comprisessaturated carbon atoms, wherein 1, 2 or 3 ring carbon atoms of the 4-,5-, 6- or 7-membered ring are optionally independently substituted with—O—, —S— or —NR^(PR)— or where 1, 2 or 3 hydrogen atoms of theheterocycle or where 1 or 2 hydrogen atoms of the 4-, 5-, 6- or7-membered ring are substituted with —OR_(PR), —SR_(PR), N(R_(PR))₂,—O—Si—(R¹³)₃, —CN, —NO₂, an ester, a thioester, a phosphoester, aphosphothioester, a phosphonoester, a phosphiniester, a sulfite ester, asulfate ester, an amide, an amino acid, a peptide, an ether, athioether, an acyl group, a thioacyl group, a carbonate, a carbamate, athioacetal, a halogen, an optionally substituted alkyl group, anoptionally substituted alkenyl group, an optionally substituted alkynylgroup, an optionally substituted aryl moiety, an optionally substitutedheteroaryl moiety, an optionally substituted monosaccharide, anoptionally substituted oligosaccharide, a nucleoside, a nucleotide, anoligonucleotide or a polymer, or,

one or more of the ring carbons are substituted with ═O or ═S,

or D comprises two 5- or 6-membered rings, wherein the rings are fusedor are linked by 1 or 2 bonds, wherein one, two or three of R⁷, R⁸ andR⁹ are not —CHR¹⁰—.

Other embodiments include a method to enhance the expression of one ormore cytokines or interleukins that facilitate Th1 immune responses in asubject or to reduce the expression of one or more cytokines orinterleukins that facilitate Th2 immune response in a subject comprisingadministering to the subject an effective amount of the composition ofclaim 32, whereby the subject's Th1 immune response is enhanced or thesubject's undesired Th2 immune response is reduced.

Embodiments include liquid formulations that comprise a formula 1compound, one or more excipients and less than about 3% water, whereinthe formulation is optionally disposed in containers that exclude water.

Another embodiment is a method comprising intermittent administration ofa formula 1 compound, to a subject having a pathological condition, suchas a viral or parasite infection.

A further embodiment is a method to modulate a subject's innateimmunity, Th1 immune responses or Th2 immune responses comprisingadministering a formula 1 compound to a subject.

Other embodiments are as described in the specification including theappended numbered embodiments and the claims.

DETAILED DESCRIPTION OF THE INVENTION

Definitions. As used herein and unless otherwise stated or implied bycontext, the following terms have the meanings defined here.

An “invention formulation” or “formulation” means an inventioncomposition that one can administer parenterally to a human or animalwithout further manipulations that change the ingredients or theingredient proportions that are present. Formulations are suitable forhuman or veterinary applications.

An “invention composition” is a composition that is an intermediate onecan use to make the invention formulations, i.e., a change(s) in aningredient(s) or its amount(s) is needed to make a formulation. Thus,invention compositions include compositions where further processing isrequired before it is a formulation, e.g., mixing or addition of adesired amount of an ingredient.

An “excipient” means a component or an ingredient that is acceptable inthe sense of being compatible with the other ingredients of inventioncompositions or formulations and not overly deleterious to the patientor animal to which the formulation is to be administered. As used here,“excipients” include liquids, such as benzyl benzoate, cottonseed oil,N,N-dimethylacetamide, a C₂₋₁₂ alcohol (e.g., ethanol), glycerol, peanutoil, a polyethylene glycol (“PEG”), vitamin E, poppyseed oil, propyleneglycol, safflower oil, sesame oil, soybean oil and vegetable oil.Excipients, as used herein will optionally exclude chloroform, dioxane,vegetable oil, DMSO or any combination of these. Excipients comprise oneor more components typically used in the pharmaceutical formulationarts, e.g., fillers, binders, disintegrants and lubricants.

A “subject” means a human or animal. Usually the animal is a vertebratesuch as a primate, rodent, domestic animal or game animal. Primatesinclude chimpanzees, cynomologous monkeys, spider monkeys, and macaques,e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbitsand hamsters. Domestic and game animals include cows, horses, pigs,deer, bison, buffalo, felines, e.g., domestic cat, canines, e.g., dog,avians, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish andsalmon. Subject includes any subset of the foregoing, e.g., all of theabove, but excluding one or more groups or species such as humans,primates or rodents.

Expressions that refer to “a formula 1 compound(s)” or “a formula 1compound” mean invention compositions or formulations where one or morethan one formula 1 compound is present, typically 1, 2, 3 or 4, usually1.

“Alkyl” as used here means linked normal, secondary, tertiary or cycliccarbon atoms, i.e., linear, branched or cyclic. The number of carbonatoms in an alkyl group or moiety is 1 to about 20, unless otherwisespecified, e.g., C₁₋₈ alkyl means an alkyl moiety containing 1, 2, 3, 4,5, 6, 7 or 8 carbon atoms. Examples include methyl, ethyl,1-propyl(n-propyl), 2-propyl(i-propyl, —CH(CH₃)₂), 1-butyl(n-butyl),2-methyl-1-propyl(i-butyl, —CH₂CH(CH₃)₂), 2-butyl(s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl(t-butyl, —C(CH₃)₃),1-pentyl(n-pentyl), 2-pentyl(—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂),2-methyl-2-butyl(—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂),3-methyl-1-butyl(—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃),1-hexyl, 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl(—CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl(—C(CH₃)₂CH₂CH₂CH₃),3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃),4-methyl-2-pentyl(—CH(CH₃)CH₂CH(CH₃)₂)3-methyl-3-pentyl(—C(CH₃)(CH₂CH₃)₂),2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂),2,3-dimethyl-2-butyl(—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl(—CH(CH₃)C(CH₃)₃), cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

“Alkenyl” means linked normal, secondary, tertiary or cyclic carbonatoms where one or more double bonds (e.g., —CH═CH—) are present,typically 1, 2 or 3, usually 1 or 2. The number of carbon atoms in analkenyl group or moiety is 2 to about 20, unless otherwise specified,e.g., C₁₋₈ alkenyl means an alkenyl moiety containing 1, 2, 3, 4, 5, 6,7 or 8 carbon atoms.

“Alkynyl” means linked normal, secondary, tertiary or cyclic carbonatoms where one or more triple bonds (—C≡C—) are present, typically 1, 2or 3, usually 1. The number of carbon atoms in an alkynyl group ormoiety is 2 to about 20, unless otherwise specified, e.g., C₁₋₈ alkynylmeans an alkynyl moiety containing 1, 2, 3, 4, 5, 6, 7 or 8 carbonatoms.

“Aryl” means phenyl or naphthyl.

“Substituted alkyl”, “substituted alkenyl” and “substituted alkynyl”mean an alkyl, alkenyl or alkynyl group that has a substituent(s) linkedto a carbon atom or substituent(s) that interrupt a carbon atom chain.Substituents include ethers (—O—), ketones (—C(O)—), —OR^(PR),—C(O)OR^(PR), —C(O)O—, —C(S)OR^(PR), —C(S)O—, —OC(O)—, —C(O)H, —OCH₂—,—OCH₂CH₂—, —OCH₂O—, —OCH₂CH₂O—, —NR^(PR)—, —N(R^(PR))₂, —NHR^(PR),—NHC(O)—, —CH₂—NR^(PR)—, —CH₂—NHR^(PR), —CH₂—NHC(O)—, —C(O)NH—,—C(O)NHR^(PR), —OC(O)NR^(PR)—, —OC(O)NHR^(PR), —NR^(PR)C(O)NR^(PR)—,—NR^(PR)C(O)NHR^(PR), —NR^(PR)CH₂—, —NR^(PR)CH₂CH₂—, —S—, —SR^(PR),—S(O)—, —S(O)(O)—, —S(O)OR^(PR), —S(O)H, —CN, —NO₂, halogen, andcombinations of these moieties where R^(PR) independently is hydrogen, aprotecting group or both R^(PR) together are a protecting group.Substituents are independently chosen when more than one is present.Alkenyl and alkynyl groups that comprise a substituent(s), are typicallysubstituted at a carbon that is one or more methylene moiety removedfrom the double bond, e.g., separated at least by one, two or more —CH₂—moieties.

Heterocycle. “Heterocycle” or “heterocyclic” includes by way of exampleand not limitation the heterocycles described in Paquette, Leo A.;“Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York,1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. 1960, 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, thiazolyl, tetrahydrothiophenyl, sulfur oxidizedtetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl,pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl,indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl,piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl,tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl,6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2dithiazinyl, thienyl, thianthrenyl,pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl,2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5—pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Typically, nitrogen bonded heterocyclesinclude 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl,and 1-piperidinyl.

“Heteroaryl” means an aromatic ring or two or more fused rings thatcontain one or more aromatic rings where the ring or fused ringscomprise 1, 2, 3 or more heteroatoms, usually oxygen (—O—), nitrogen(—NX—) or sulfur (—S—) where X is —H, a protecting group or C₁₋₆ alkyl,usually —H. Examples are as described for heterocycle.

“Alcohol” as used herein, usually in the context of excipients, means analcohol that comprises a C₂₋₁₂ alkyl moiety substituted at a hydrogenatom with one hydroxyl group. Alcohols include ethanol, n-propanol,i-propanol, n-butanol, i-butanol, s-butanol, t-butanol, n-pentanol,i-pentanol, n-hexanol, cyclohexanol, n-heptanol, n-octanol, n-nonanoland n-decanol. The carbon atoms in alcohols can be straight, branched orcyclic. Alcohol includes any subset of the foregoing, e.g., C₂₋₄alcohols (alcohols having 2, 3 or 4 carbon atoms).

“Halogen” means fluorine, chlorine, bromine or iodine.

“Protecting group” means a moiety that prevents the atom to which it islinked from participating in unwanted reactions. For example, for—OR^(PR), R^(PR) may be hydrogen or a protecting group for the oxygenatom found in a hydroxyl, while for —C(O)—OR^(PR), R^(PR) may behydrogen or a carboxyl protecting group, for —SR^(PR), R^(PR) may behydrogen or a protecting group for sulfur in thiols for instance, andfor —NHR^(PR) or —N(R^(PR))₂—, R^(PR) may be hydrogen or a nitrogen atomprotecting group for primary or secondary amines. Hydroxyl, amine andother reactive groups are found in formula 1 compounds at, e.g., R¹ orR². These groups may require protection against reactions taking placeelsewhere in the molecule. The protecting groups for oxygen, sulfur ornitrogen atoms are usually used to prevent unwanted reactions withelectrophilic compounds, such as acylating used, e.g., in steroidchemistry.

“Ester” means a moiety that comprises a —C(O)—O— structure. Typically,esters as used here comprise an organic moiety containing about 1-50carbon atoms (e.g., about 2-20 carbon atoms) and 0 to about 10independently selected heteroatoms (e.g., O, S, N, P, Si), where theorganic moiety is bonded to a formula 1 steroid nucleus at, e.g., R¹ orR² through the —C(O)—O— structure, e.g., organic moiety-C(O)—O-steroidor organic moiety-O—C(O)-steroid. The organic moiety usually comprisesone or more of any of the organic groups described above, e.g., C₁₋₂₀alkyl moieties, C₂₋₂₀ alkenyl moieties, C₂₋₂₀ alkynyl moieties, arylmoieties, C₂₋₉ heterocycles or substituted derivatives of any of these,e.g., comprising 1, 2, 3, 4 or more substituents, where each substituentis independently chosen. Typical substitutions for hydrogen or carbonatoms in these organic groups include 1, 2, 3, 4 or more, usually 1, 2,or 3-O—, —S—, —NR^(PR)— (including —NH—), —C(O)—, ═O, ═S, —N(R^(PR))₂(including —NH₂), —C(O)OR^(PR) (including —C(O)OH), —OC(O)R^(PR)(including —O—C(O)—H), —OR^(PR) (including —OH), —SR^(PR) (including—SH), —NO₂, —CN, —NHC(O)—, —C(O)NH—, —OC(O)—, —C(O)O—, —O-A8, —S-A8,—C(O)-A8, —OC(O)-A8, —C(O)O-A8, ═N—, —N═, ═N—OH, —OPO₃(R^(PR))₂, —OSO₃H₂or halogen moieties or atoms, where each R^(PR) is —H, an independentlyselected protecting group or both R_(PR) together comprise a protectinggroup, and A8 is C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, C₁₋₄ alkyl-aryl(e.g., benzyl), aryl (e.g. phenyl) or C₀₋₄ alkyl-C₂₋₉ heterocycle.Substitutions are independently chosen. The organic moiety includescompounds defined by the R₄ variable. The organic moieties excludeobviously unstable moieties, e.g., —O—O—, except where such unstablemoieties are transient species that one can use to make a compound withsufficient chemical stability for one or more of the uses describedherein. The substitutions listed above are typically substituents thatone can use to replace one or more carbon atoms, e.g., —O— or —C(O)—, orone or more hydrogen atom, e.g., halogen, —NH₂ or —OH.

“Thioester” means a moiety that comprises a —C(S)—O— structure.Typically, thioesters as used here comprise an organic moiety containingabout 1-50 carbon atoms (e.g., about 2-20 carbon atoms) and 0 to about10 heteroatoms (e.g., O, S, N, P, Si), where the organic moiety isbonded to a formula 1 steroid nucleus at R² through the —C(S)—O—structure, e.g., organic moiety-C(S)—O-steroid or organicmoiety-O—C(S)-steroid. The organic moiety is as described above foresters.

“Thioacetal” means a moiety that comprises a —C(O)—S— structure.Typically, thioacetals as used here comprise an organic moietycontaining about 1-50 carbon atoms (e.g., about 2-20 carbon atoms) and 0to about 10 heteroatoms (e.g., O, S, N, P, Si), where the organic moietyis bonded to a formula 1 steroid nucleus at R² through the —C(O)—S—structure, e.g., organic moiety-C(O)—S-steroid or organicmoiety-S—C(O)-steroid. The organic moiety is as described above foresters.

“Phosphoester” or “phosphate ester” means a moiety that comprises a—O—P(OR^(PR))(O)—O— structure where R^(PR) is hydrogen (—H), aprotecting group or an organic moiety as described for esters.Typically, phosphoesters as used here comprise a hydrogen atom, aprotecting group or an organic moiety containing about 1-50 carbon atomsand 0 to about 10 heteroatoms (e.g., O, S, N, P, Si) linked to a formula1 steroid nucleus at R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸ through the—O—P(O)(O)—O— structure, e.g., organic moiety-O—P(O)(OH)—O-steroid. Theorganic moiety is as described above for esters.

“Phosphothioester” means a moiety that comprises a —O—P(SR^(PR))(O)—O—structure where R^(PR) is —H, a protecting group or an organic moiety asdescribed for esters. Typically, phosphothioesters as used here comprisea hydrogen atom, a protecting group or an organic moiety containingabout 1-50 carbon atoms and 0 to about 10 heteroatoms (e.g., O, S, N, P,Si) linked to a formula 1 steroid nucleus at R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸through the —O—P(O)(O)—O— structure, e.g., organicmoiety-O—P(O)(SH)—O-steroid. The organic moiety is as described abovefor esters.

“Phosphonoester” means a moiety that comprises a —P(OR^(PR))(O)—O—structure where R^(PR) is —H, a protecting group or an organic moiety asdescribed for esters. Typically, phosphonoesters as used here comprise ahydrogen atom, a protecting group or an organic moiety containing about1-50 carbon atoms and 0 to about 10 heteroatoms (e.g., O, S, N, P, Si)linked to a formula 1 steroid nucleus at R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸through the —P(OR^(PR))(O)—O— structure, i.e., organicmoiety-P(OR^(PR))(O)—O-steroid or steroid-P(OR^(PR))(O)—O-organicmoiety. The organic moiety is as described above for esters.

“Phosphiniester” means a moiety that comprises a —P(OR^(PR))—O—structure where R^(PR) is —H, a protecting group or an organic moiety asdescribed for esters. Typically, phosphiniesters as used here comprise ahydrogen atom, a protecting group or an organic moiety containing about1-50 carbon atoms and 0 to about 10 heteroatoms (e.g., O, S, N, P, Si)linked to a formula 1 steroid nucleus at R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸through the —P(OR^(PR))—O— structure, i.e., organic moiety-P(OR^(PR))—O—steroid or steroid-P(OR^(PR))—O-organic moiety. The organic moiety is asdescribed above for esters.

“Sulfate ester” means a moiety that comprises a —O—S(O)(O)—O— structure.Typically, sulfate esters as used here comprise a hydrogen atom, aprotecting group or an organic moiety containing about 1-50 carbon atomsand 0 to about 10 heteroatoms (e.g., O, S, N, P, Si) linked to a formula1 steroid nucleus at R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸ through the—O—S(O)(O)—O— structure, e.g., organic moiety-O—S(O)(O)—O-steroid. Theorganic moiety is as described above for esters.

“Sulfite ester” means a moiety that comprises a —O—S(O)—O— structure.Typically, sulfite esters as used here comprise an organic moietycontaining about 1-50 carbon atoms and 0 to about 10 heteroatoms (e.g.,O, S, N, P, Si) linked to a formula 1 steroid nucleus at R¹-R⁶, R¹⁰,R¹⁵, R¹⁷ or R¹⁸ through the —O—S(O)—O— structure, e.g., organicmoiety-O—S(O)—O-steroid. The organic moiety is as described above foresters.

“Thioacetal” means a moiety that comprises a —S—C(O)— structure.Typically, thioacetal groups as used here comprise an organic moietycontaining about 1-50 carbon atoms and 0 to about 10 heteroatoms (e.g.,O, S, N, P, Si) linked to a formula 1 steroid nucleus at R¹-R⁶, R¹⁰,R¹⁵, R¹⁷ or R¹⁸ through the —S—C(O)— structure e.g., organicmoiety-S—C(O)-steroid or steroid-S—C(O)-organic moiety. The organicmoiety is as described above for esters.

“Amide” means an organic moiety as described for ester that comprises 1,2, 3, 4 or more —C(O)—NR^(PR)— moieties, usually 1 or 2, where R^(PR) is—H or a protecting group, R^(PR) is usually H. In some embodiments, the—C(O)NR^(PR)— group is linked to the steroid nucleus at R¹-R⁶, R¹⁰, R¹⁵,R¹⁷ or R¹⁸, i.e., organic moiety-C(O)NR^(PR)-steroid orsteroid-C(O)NR^(PR)-organic moiety.

“Ether” means an organic moiety as described for ester that comprises 1,2, 3, 4 or more —O— moieties, usually 1 or 2. In some embodiments, the—O— group is linked to the steroid nucleus at R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ orR¹⁸, e.g., organic moiety-O-steroid.

“Thioether” means an organic moiety as described for ester thatcomprises 1, 2, 3, 4 or more —S— moieties, usually 1 or 2. In someembodiments, the —S-group is linked to the steroid nucleus at R¹-R⁶,R¹⁰, R¹⁵, R¹⁷ or R¹⁸, e.g., organic moiety-S-steroid.

“Acyl group” means an organic moiety as described for ester thatcomprises 1, 2, 3, 4 or more —C(O)— groups. In some embodiments, the—C(O)— group is linked to the steroid nucleus at R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ orR¹⁸, e.g., organic moiety-C(O)-steroid.

“Thioacyl” means an organic moiety as described for ester that comprises1, 2, 3, 4 or more —C(S)— groups. In some embodiments, the —C(S)— groupis linked to the steroid nucleus at R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸, e.g.,organic moiety-C(S)-steroid.

“Carbonate” means an organic moiety as described for ester thatcomprises 1, 2, 3, 4 or more —O—C(O)—O— structures. Typically, carbonategroups as used here comprise an organic moiety containing about 1-50carbon atoms and 0 to about 10 heteroatoms (e.g., O, S, N, P, Si) linkedto a formula 1 steroid nucleus at R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ or R¹⁸ throughthe —O—C(O)—O— structure, e.g., organic moiety-O—C(O)—O-steroid.

“Carbamate” means an organic moiety as described for ester thatcomprises 1, 2, 3, 4 or more —O—C(O)NR^(PR)— structures where R^(PR) is—H, a protecting group or an organic moiety as described for ester.Typically, carbamate groups as used here comprise an organic moietycontaining about 1-50 carbon atoms and 0 to about 10 heteroatoms (e.g.,O, S, N, P, Si) linked to a formula 1 steroid nucleus at R¹-R⁶, R¹⁰,R¹⁵, R⁷ or R¹⁸ through the —O—C(O)—NR^(PR)— structure, e.g., organicmoiety-O—C(O)—NR^(PR)— steroid or steroid-O—C(O)—NR^(PR)-organic moiety.

As used herein, “monosaccharide” means a polyhydroxy aldehyde or ketonehaving the empirical formula (CH₂O)_(n) where n is 3, 4, 5, 6 or 7.Monosaccharide includes open chain and closed chain forms, but willusually be closed chain forms. Monosaccharide includes hexofuranose andpentofuranose sugars such as 2′-deoxyribose, ribose, arabinose, xylose,their 2′-deoxy and 3′-deoxy derivatives and their 2′,3′-dideoxyderivatives. Monosaccharide also includes the 2′,3′ dideoxydidehydroderivative of ribose. Monosaccharides include the D-, L- and DL-isomersof glucose, fructose, mannose, idose, galactose, allose, gulose,altrose, talose, fucose, erythrose, threose, lyxose, erythrulose,ribulose, xylulose, ribose, arabinose, xylose, psicose, sorbose,tagatose, glyceraldehyde, dihydroxyacetone and their monodeoxyderivatives such as rhamnose. Monosaccharides are optionally protectedor partially protected.

Optionally substituted alkyl group, optionally substituted alkenylgroup, optionally substituted alkynyl group, optionally substituted arylmoiety and optionally substituted heterocycle mean substitutions thatinclude C₁₋₂₀ alkyl moieties, C₂₋₂₀ alkenyl moieties, C₂₋₂₀ alkynylmoieties, aryl moieties, C₂₋₉ heterocycles or substituted derivatives ofany of these. Typical substitutions for these organic groups include 1,2, 3, 4 or more, usually 1 or 2, —O—, —S—, —NR^(PR)—, —C(O)—,—N(R^(PR))₂, —C(O)OR^(PR), —OC(O)R^(PR), —OR^(PR), —SR^(PR), —NO₂, —CN,—NHC(O)—, —C(O)NH—, —OC(O)—, —C(O)O—, —O-A8, —S-A8, —C(O)-A8, —OC(O)-A8,—C(O)O-A8, ═N—, —N═, —OPO₂R^(PR), —OSO₃H or halogen moieties or atoms,where R^(PR) independently is —H, a protecting group or both R^(PR)together are a protecting group and A8 is C₁₋₈ alkyl, C₁₋₈ alkenyl, C₁₋₈alkynyl, C₁₋₄ alkyl-aryl (e.g., benzyl), aryl (e.g. phenyl) or C₁₋₄alkyl-C₁₋₅ heterocycle. Substitutions are independently chosen. Theorganic moieties as described here, and for other any other moietiesdescribed herein, exclude obviously unstable moieties, e.g., —O—O—,except where such unstable moieties are transient species that one canuse to make a compound with sufficient chemical stability for the one ormore of the uses described herein.

Optionally substituted “monosaccharide” comprise any C3-C7 sugar, D-, L-or DL-configurations, e.g., erythrose, glycerol, ribose, deoxyribose,arabinose, glucose, mannose, galactose, fucose, mannose, glucosamine,N-acetylneuraminic acid, N-acetylglucosamine, N-acetylgalactosamine thatis optionally substituted at one or more hydroxyl groups. Suitablesubstitutions include hydrogen, protected hydroxyl, carboxyl, azido,cyano, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —O—C₂₋₆ alkenyl, —S—C₂₋₆ alkenyl,optionally protected amine, optionally protected carboxyl, halogen,thiol or protected thiol. The linkage between the monosaccharide thesteroid is α or β.

Optionally substituted “oligosaccharide” comprises two, three, four ormore of any C3-C7 sugars that are covalently linked to each other. Thelinked sugars may have D-, L- or DL-configurations. Suitable sugars andsubstitutions are as described for monosaccharides. The linkage betweenthe oligosaccharide and the steroid is α or β, as are the linkagesbetween the monosaccharides that comprise the oligosaccharide.

Nucleoside includes 3TC, AZT, D4T, ddI, ddC, G, A, U, C, T, dG, dA, dTand dC.

Polymer includes biocompatible organic polymers, e.g., PEGs andpolyhydroxyalkyl polymers.

PEG means an ethylene glycol polymer that contains about 20 to about2000000 linked monomers, typically about 50-1000 linked monomers,usually about 100-300. Polyethylene glycols include PEGs containingvarious numbers of linked monomers, e.g., PEG20, PEG30, PEG40, PEG60,PEG80, PEG100, PEG115, PEG 200, PEG 300, PEG400, PEG500, PEG600, PEG1000, PEG1500, PEG2000, PEG 3350, PEG4000, PEG4600, PEG5000, PEG6000,PEG8000, PEG11000, PEG12000, PEG2000000 and any mixtures thereof.

Amino acid. “Amino acid” means an amino acid moiety that comprises anynaturally-occurring or synthetic amino acid residue, i.e., any moietycomprising at least one carboxyl and at least one amino residue directlylinked by one, two three or more carbon atoms, typically one (α) carbonatom. The nature and identity of the intervening structure locatedbetween the carboxyl and amino groups can have a variety of structuresincluding those described herein. Typically, amino acids linked to thesteroid through the amine group have sufficient conformation and lengthto be capable of autocatalytic hydrolysis of the amino acid-steroid bondand release of the steroid. This can occur when the free carboxyl isgenerated in vivo by deesterification, deamidation or peptidolyticcleavage of the precursor containing a linkage between the amino acid'samine group and the steroid. Hydrolysis of the bond between an aminoacid's carboxyl or amino group and the steroid can also occur bychemical or enzymatic activity, e.g., esterase cleavage or non-enzymatichydrolysis.

In general, the amino acids corresponding to the residues employed inthe invention compounds are naturally occurring and have no significantpharmacological activity per se. However, optimal pharmacokineticactivity, (substantially complete hydrolysis upon hydrolysis of thedistal amide or ester bond) may be achieved by using non-naturallyoccurring amino acid residues. The intervening structure may be assimple as methylene when the amino acid residue is glycyl, orsubstituted methylene for other a amino acids. The structure ordinarilycontains up to about 5 carbon or heteroatoms in the direct linkagebetween the amino acid's carboxyl carbon and the amine nitrogen. Thus,amino acids can comprise intervening ethylene, propylene, butylene, orpentylene groups or their substituted analogs, such as for example,oxyesters or ethers in which oxygen replaces carbon and, as appropriate,hydrogen. An example of such an intervening structure would be—CH—O—C(R²²)(R²³)—, where R²² and R²³ are independently selectedhydrogen or organic moieties as described above for esters. In someembodiments one of R²² and R²³ is hydrogen and the other is a C2-20organic moiety. Typically the organic moieties contain about 1-20 carbonatoms and 0, 1, 2, 3, 4 or 5 independently selected heteroatoms, whichare typically selected from oxygen, nitrogen, sulfur and phosphorus. Ingeneral, fewer intervening atoms are used when more rapid hydrolysis isdesired, although larger structures are suitable if, e.g., they possesssufficient flexibility or have conformations to allow positioning of thecarboxyl group in proximity to the amino acid-steroid bond.

Ordinarily, R²² is —H, methyl or hydroxymethyl, usually —H, and R²³ is aside chain or group of a naturally occurring amino acid. Amino acid sidechains include analogs where the side chain is a C₁₋₁₅ homolog of thecorresponding natural compound, e.g., methylene, ethylene, propylene,butylene or a substituted derivative thereof, e.g., an alkyl, ether oralkoxy (e.g., methoxy, ethoxy, propoxy) substituted derivative. Ingeneral, for carboxyl-containing side chains, if the C atom of the sidechain carboxyl is linked by 5 or less atoms to the N then the carboxyloptionally will be blocked, e.g. by esterification or amidation whereinthe ester or amide bonds are hydrolyzable in vivo. R²² also is takentogether with R³⁰ to form a proline residue (—CH₂—)₃. Thus, R²³ isgenerally a side group such as —H, —CH₃, —CH(CH₃)₂, —CH₂—CH(CH₃)₂,—CHCH₃—CH₂—CH₃, —CH₂—C₆H₅, —CH₂CH₂—S—CH₃, —CH₂OH, —CH(OH)—CH₃, —CH₂—SH,—CH₂—C₆H₄OH, —CH₂—CO—NH₂, —CH₂—CH₂—CO—NH₂, —CH₂—COOH, CH₂—CH₂—COOH,—(CH₂)₄—NH₂ and —(CH₂)₃—NH—C(NH₂)—NH₂. R²³ also includes1-guanidinoprop-3-yl, benzyl, 4-hydroxybenzyl, imidazol-4-yl,indol-3-yl, methoxyphenyl and ethoxyphenyl. The optimal R³⁰ group isreadily selected using routine assays.

In general, the amino acid residue has the structure shown in theformulas below. Ordinarily, n is 1 or 2, R²² is —H and R²³ is a moietycontaining one or more of the following groups: amino, carboxyl, amide,carboxyl ester, hydroxyl, C₆-C₇ aryl, ether (—O—), thioether (—S—), n-,s- or t-alkyl (C₁-C₆), guanidinyl, imidazolyl, indolyl, sulfhydryl,sulfoxide, and phosphoryl. The R²² and R²³ substituents can have a widevariety of structures including those disclosed herein, e.g., esters,ethers or carbonates.

When the amino acid residues contain one or more chiral centers, any ofthe D, L, meso, threo or erythro (as appropriate) racemates or mixturesthereof, fall within the scope of this invention. In general, if it isdesired to rely on non-enzymatic means of hydrolysis, D isomers shouldbe used. On the other hand, L isomers may be more versatile since theycan be susceptible to both non-enzymatic as well as potential targetedenzymatic hydrolysis, and are more efficiently transported by amino acidor dipeptidyl transport systems in the gastrointestinal tract.

Examples of suitable amino acid residues include the following: Glycyl;aminopolycarboxylic acids, e.g., aspartic acid, β-hydroxyaspartic acid,glutamic acid, β-hydroxyglutamic acid, β-methylaspartic acid,β-methylglutamic acid, β,β-dimethylaspartic acid, γ-hydroxyglutamicacid, β,γ-dihydroxyglutamic acid, β-phenylglutamic acid,γ-methyleneglutamic acid, 3-aminoadipic acid, 2-aminopimelic acid,2-aminosuberic acid and 2-aminosebacic acid residues; amino acid amidessuch as glutaminyl and asparaginyl; polyamino- orpolybasic-monocarboxylic acids such as arginine, lysine, β-aminoalanine,γ-aminobutyrine, ornithine, citruline, homoarginine, homocitrulline,5-hydroxy-2,6-diaminohexanoic acid (commonly, hydroxylysine, includingallohydroxylysine) and diaminobutyric acid residues; other basic aminoacid residues such as histidinyl; diaminodicarboxylic acids such asα,α′-diaminosuccinic acid, α,α′-diaminoglutaric acid, α,α′-diaminoadipicacid, α,α′-diaminopimelic acid, α,α′-diamino-β-hydroxypimelic acid,α,α′-diaminosuberic acid, α,α′-diaminoazelaic acid, andα,α′-diaminosebacic acid residues; imino acids such as proline, 4- or3-hydroxy-2-pyrrolidinecarboxylic acid (commonly, hydroxyproline,including allohydroxyproline), γ-methylproline, pipecolic acid,5-hydroxypipecolic acid, —N([CH₂]_(n)COOR^(PR))₂, wherein n is 1, 2, 3,4, 5 or 6 and R^(PR) is —H or a protecting group, andazetidine-2-carboxylic acid residues; a mono- or di-alkyl (typicallyC₁-C₈ branched or normal) amino acid such as alanine, valine, leucine,allylglycine, butyrine, norvaline, norleucine, heptyline,α-methylserine, α-amino-α-methyl-γ-hydroxyvaleric acid,α-amino-α-methyl-δ-hydroxyvaleric acid,α-amino-α-methyl-ε-hydroxycaproic acid, isovaline, α-methylglutamicacid, α-aminoisobutyric acid, α-aminodiethylacetic acid,α-aminodiisopropylacetic acid, α-aminodi-n-propylacetic acid,α-aminodiisobutylacetic acid, α-aminodi-n-butylacetic acid,α-aminoethylisopropylacetic acid, α-amino-n-propylacetic acid,α-aminodiisoamyacetic acid, α-methylaspartic acid, α-methylglutamicacid, 1-aminocyclopropane-1-carboxylic acid; isoleucine, alloisoleucine,tert-leucine, β-methyltryptophan and α-amino-β-ethyl-β-phenylpropionicacid residues; β-phenylserinyl; aliphatic α-amino-β-hydroxy acids suchas serine, β-hydroxyleucine, β-hydroxynorleucine, β-hydroxynorvaline,and α-amino-β-hydroxystearic acid residues; α-Amino, α-, γ-, δ- orε-hydroxy acids such as homoserine, γ-hydroxynorvaline,δ-hydroxynorvaline and epsilon-hydroxynorleucine residues; canavinyl andcanalinyl; γ-hydroxyornithinyl; 2-Hexosaminic acids such asD-glucosaminic acid or D-galactosaminic acid residues; α-amino-β-thiolssuch as penicillamine, β-thiolnorvaline or β-thiolbutyrine residues;other sulfur containing amino acid residues including cysteine;homocystine; β-phenylmethionine; methionine; S-allyl-L-cysteinesulfoxide; 2-thiolhistidine; cystathionine; and thiol ethers of cysteineor homocysteine; phenylalanine, tryptophan and ring-substituted α aminoacids such as the phenyl- or cyclohexylamino acids α-aminophenylaceticacid, α-aminocyclohexylacetic acid and α-amino-β-cyclohexylpropionicacid; phenylalanine analogues and derivatives comprising aryl, loweralkyl, hydroxy, guanidino, oxyalkylether, nitro, sulfur orhalo-substituted phenyl (e.g., tyrosine, methyltyrosine and o-chloro-,p-chloro-, 3,4-dicloro, o-, m- or p-methyl-, 2,4,6-trimethyl-,2-ethoxy-5-nitro, 2-hydroxy-5-nitro and p-nitro-phenylalanine); furyl-,thienyl-, pyridyl-, pyrimidinyl-, purine or naphthylalanines; andtryptophan analogues and derivatives including kynurenine,3-hydroxykynurenine, 2-hydroxytryptophan and 4-carboxytryptophanresidues; α-amino substituted amino acid residues including sarcosine(N-methylglycine), N-benzylglycine, N-methylalanine, N-benzylalanine,N-methylphenylalanine, N-benzylphenylalanine, N-methylvaline andN-benzylvaline; and α-Hydroxy and substituted α-hydroxy amino acidresidues including serine, threonine, allothreonine, phosphoserine andphosphothreonine residues.

Any one of the foregoing or other known amino acids are suitablyemployed in this invention. Typically amino acids are capable ofautocatalytically hydrolyzing the amino acid-steroid bond. Thus, theytypically contain, or upon being hydrolyzed in vivo, contain a freecarboxyl group or amine group.

Also of interest are hydrophobic amino acids such as mono-or di-alkyl oraryl amino acids, cycloalkylamino acids and the like. These residues,together with R²⁹-R³⁴ (R³¹-R³⁴ are defined below) can contribute to cellpermeability by modulating the lipophilicity of a formula 1 or formula 2compound. Typically, the residue does not contain a sulfhydryl orguanidino substituent.

Peptide. One, 2, 3 or more of R¹-R⁴ can comprise a “peptide”, i.e., twoor more amino acids as defined above. Typically the amino acids arelinked through normal peptide bonds, i.e., —CO—NH—, between adjacentamino acid residues. Peptides comprise dipeptides (dimers), tripeptides(trimers), short peptides of 4, 5, 6, 8, 10 or 15 residues, and longerpeptides or proteins having about 100 or more residues. Inventioncompounds that comprise a peptide can be used as immunogens, prodrugs oras synthetic precursors for other steroid derivatives. In oneembodiment, the peptide will contain a peptidolytic enzyme cleavage siteat the peptide bond linking the first residue and the next residuedistal to the steroid residue. Such cleavage sites are optionallyflanked by enzymatic recognition structures, e.g. particular residuesrecognized by a hydrolytic enzyme, e.g., a peptidase located in theserum or in cells.

Peptidolytic enzymes are well known, and in particular includecarboxypeptidases. Carboxypeptidases digest polypeptides by removingC-terminal residues, and are specific in many instances for particularC-terminal sequences. Such enzymes and their substrate requirements ingeneral are well known. For example, a dipeptide having a given pair ofresidues and a free carboxyl terminus is covalently bonded through itsα-amino group to the steroid nucleus. It is expected that the peptidewill be cleaved by the appropriate dipeptidase, protease or by chemicalhydrolysis, leaving the carboxyl of the proximal amino acid residue toautocatalytically cleave the amidate bond.

Examples of suitable dipeptidyl groups (designated by their singleletter symbols) are shown in the table below.

SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr tyrosine G Gly glycine F Phephenylalanine M Met methionine A Ala alanine S Ser serine I Ileisoleucine L Leu leucine T Thr threonine V Val valine P Pro proline KLys lysine H His histidine Q Gln glutamine E Glu glutamic acid W Trptryptophan R Arg arginine D Asp aspartic acid N Asn asparagine C Cyscysteine Dipeptides AA, AR, AN, AD, AC, AE, AQ, AG, AH, AI, AL, AK, AM,AF, AP, AS, AT, AW, AY, AV, RA, RR, RN, RD, RC, RE, RQ, RG, RH, RI, RL,RK, RM, RF, RP, RS, RT, RW, RY, RV, NA, NR, NN, ND, NC, NE, NQ, NG, NH,NI, NL, NK, NM, NF, NP, NS, NT, NW, NY, NV, DA, DR, DN, DD, DC, DE, DQ,DG, DH, DI, DL, DK, DM, DF, DP, DS, DT, DW, DY, DV, CA, CR, CN, CD, CC,CE, CQ, CG, CH, CI, CL, CK, CM, CF, CP, CS, CT, CW, CY, CV, EA, ER, EN,ED, EC, EE, EQ, EG, EH, EI, EL, EK, EM, EF, EP, ES, ET, EW, EY, EV, QA,QR, QN, QD, QC, QE, QQ, QG, QH, QI, QL, QK, QM, QF, QP, QS, QT, QW, QY,QV, GA, GR, GN, GD, GC, GE, GQ, GG, GH, GI, GL, GK, GM, GF, GP, GS, GT,GW, GY, GV, HA, HR, HN, HD, HC, HE, HQ, HG, HH, HI, HL, HK, HM, HF, HP,HS, HT, HW, HY, HV, IA, IR, IN, ID, IC, IE, IQ, IG, IH, II, IL, IK, IM,IF, IP, IS, IT, IW, IY, IV, LA, LR, LN, LD, LC, LE, LQ, LG, LH, LI, LL,LK, LM, LF, LP, LS, LT, LW, LY, LV, KA, KR, KN, KD, KC, KE, KQ, KG, KH,KI, KL, KK, KM, KF, KP, KS, KT, KW, KY, KV, MA, MR, MN, MD, MC, ME, MQ,MG, MH, MI, ML, MK, MM, MF, MP, MS, MT, MW, MY, MV, FA, FR, FN, FD, FC,FE, FQ, FG, FH, FI, FL, FK, FM, FF, FP, FS, FT, FW, FY, FV, PA, PR, PN,PD, PC, PE, PQ, PG, PH, PI, PL, PK, PM, PF, PP, PS, PT, PW, PY, PV, SA,SR, SN, SD, SC, SE, SQ, SG, SH, SI, SL, SK, SM, SF, SP, SS, ST, SW, SY,SV, TA, TR, TN, TD, TC, TE, TQ, TG, TH, TI, TL, TK, TM, TF, TP, TS, TT,TW, TY, TV, WA, WR, WN, WD, WC, WE, WQ, WG, WH, WI, WL, WK, WM, WF, WP,WS, WT, WW, WY, WV, YA, YR, YN, YD, YC, YE, YQ, YG, YH, YI, YL, YK, YM,YF, YP, YS, YT, YW, YY, YV, VA, VR, VN, VD, VC, VE, VQ, VG, VH, VI, VL,VK, VM, VF, VP, VS, VT, VW, VY, VV

Such dipeptides include species where both amino acids are in the Lconfiguration, the D configuration or mixtures of configurations.

Tripeptides, i.e., 3 linked amino acid residues, are also usefulembodiments. Tripeptides include those where A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, W or Y is linked by a standard peptide bondto the amino or the carboxyl terminus of any of the dipeptides listedabove. The sequence -X1-pro-X2- (where X1 is any amino acid and X2 ishydrogen, any amino acid residue or a carboxyl ester of proline) will becleaved by luminal carboxypeptidase to yield X1 with a free carboxyl,which in turn autocatalytically cleaves the amidate bond. X2 usuallywill be a benzyl ester of the carboxy group of X2. Other embodimentsinclude tetrapeptides such as ones where any two of the dipeptideslisted above, which may be the same or different dipeptides (e.g., AAand M linked together or, e.g., M and GI linked together), are linked toeach other by a peptide bond through the amino terminus or carboxylterminus. One, 2 or more tetrapeptides may bonded to the formula 1 orformula 2 compound through the tetrapeptide's amino or carboxylterminus.

In some embodiments, the formula 1 or formula 2 compound comprises oneor more amino acids or peptides having the structure (A), (B) or (C):(A)R³²—NH—{[C(R²⁹)(R³⁰)]_(b)—C(O)—N(R³¹)}_(f)—[C(R²⁹)(R³⁰)]_(a)—C(O)—O-steroid,(B)R³³—O—{C(O)—[C(R²⁹)(R³⁰)]_(d)—N(R³¹)}_(g)—C(O)—[C(R²⁹)(R³⁰)]_(c)—N(R³¹)—O-steroid,or (C)R³³—O—{C(O)—[C(R²⁹)(R³⁰)]_(d)—N(R³¹)}_(e)—C(O)—[C(R²⁹)(R³⁰)]_(c)—N(R³¹)—C(O)—O-steroid,wherein (A), (B) or (C) are independently selected and they are bondedto 1, 2, 3 or more of R¹ through R⁴, where each R²⁹-R³¹ is independentlyselected; R²⁹ independently are —H or a C1-20 organic moiety (e.g., C₁₋₆alkyl, e.g. —CH₃ or —C₂H₅); R³⁰ independently are the side chain of anamino acid, including the side chain of naturally occurring amino acidsas described above, e.g., —H, —CH₃, —CH₂C₆H₅; R³¹ is —H or a protectinggroup; R³² and R³³ independently comprise —H, a protecting group, anester or an amide where each atom or group is independently chosen; a,b, c and d independently are 1, 2, 3, 4 or 5, usually 1; e, f and gindependently are an integer from 0 to about 1000, typically theyindependently are 0, 1, 2, 3, 4, 5, 6, 7 or 8; a, b, c and dindependently are 1 or 2; e, f and g independently are 0, 1, 2, 3, 4 or5.

If the amino acid(s) or residue(s) has 2 or more amine groups, e.g., alysinyl or arginyl, or ornithinyl residue, then R²⁹ is usually —H andR³⁰ may comprise —[C(R³⁴)₂]_(n2)N(R^(PR))— where n2 is 0, 1, 2, 3, 4, 5or 6, R^(PR) is —H or a protecting g each R³⁴ independently is —H,C₁-C₂₀ optionally substituted alkyl, C₆-C₂₀ optionally substituted aryl,C₇-C₂₀ optionally substituted alkylaryl, C₇-C₂₀ optionally substitutedarylalkyl, C₁-C₂₀ optionally substituted alkoxy, C₆-C₂₀ optionallysubstituted aryloxy or hydroxyl. Such compounds will contain a pluralityof steroid moieties. For example when both the epsilon (ε) or delta (δ)and alpha (α) amino groups of lysine or ornithine are substituted withsteroid moieties the amidate is believed to be capable of releasing twomolecules of active drug, each expected to emerge under differentpharmacokinetics and therefore further sustaining the drug release.

Salt. Invention embodiments include salts and complexes of inventioncompounds (formula 1 compounds), including pharmaceutically acceptableor salts that are relatively non-toxic. Some of the invention compoundshave one or more moieties that carry at least a partial positive ornegative charge in aqueous solutions, typically at a pH of about 4-10,that can participate in forming a salt, a complex, a composition withpartial salt and partial complex properties or other noncovalentinteractions, all of which we refer to as a “salt(s)”. Salts are usuallybiologically compatible or pharmaceutically acceptable or non-toxic,particularly for mammalian cells. Salts that are biologically toxic areoptionally used with synthetic intermediates of invention compounds.When a water-soluble composition is desired, monovalent salts areusually used.

Metal salts typically are prepared by reacting the metal hydroxide witha compound of this invention. Examples of metal salts that areoptionally prepared in this way are salts containing Li⁺, Na⁺, and K⁺. Aless soluble metal salt can be precipitated from the solution of a moresoluble salt by adding a suitable metal compound. Invention salts may beformed from acid addition of certain organic acids, such as organiccarboxylic acids, and inorganic acids, such as alkylsulfonic acids orhydrogen halide acids, to acidic or basic centers on inventioncompounds, such as basic centers on the invention pyrimidine baseanalogs. Metal salts include ones containing Na⁺, Li⁺, K⁺, Ca⁺⁺ or Mg⁺⁺.Other metal salts may contain aluminum, barium, strontium, cadmium,bismuth, arsenic or zinc ion.

Salt(s) of invention compounds may comprise a combination of appropriatecations such as alkali and alkaline earth metal ions or ammonium andquaternary ammonium ions with the acid anion moiety of the phosphoricacid or phosphonic acid group, which may be present in inventionpolymers or monomers.

Salts are produced by standard methods, including dissolving free basein an aqueous, aqueous-alcohol or aqueous-organic solution containingthe selected acid, optionally followed by evaporating the solution. Thefree base is reacted in an organic solution containing the acid, inwhich case the salt usually separates directly or one can concentratethe solution.

Suitable amine salts include amines having sufficient basicity to form astable salt, usually amines of low toxicity including trialkyl amines(tripropylamine, triethylamine, trimethylamine), procaine,dibenzylamine, N-benzyl-betaphenethylamine, ephenamine,N,N′-dibenzylethylenediamine, N-ethylpiperidine, benzylamine anddicyclohexylamine.

Salts include organic sulfonic acid or organic carboxylic acid salts,made for example by addition of the acids to basic centers, typicallyamines. Exemplary sulfonic acids include C₆₋₁₆ aryl sulfonic acids,C₆₋₁₆ heteroaryl sulfonic acids and C₁₋₁₆ alkyl sulfonic acids such asphenyl sulfonic acid, a-naphthalene sulfonic acid, β-naphthalenesulfonic acid, (S)-camphorsulfonic acid, methyl (CH₃SO₃H), ethyl(C₂H₅SO₃H), n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl,pentyl and hexyl sulfonic acids. Exemplary organic carboxylic acidsinclude C₁₋₁₆ alkyl, C₆₋₁₆ aryl carboxylic acids and C₄₋₁₆ heteroarylcarboxylic acids such as acetic, glycolic, lactic, pyruvic, malonic,glutaric, tartaric, citric, fumaric, succinic, malic, maleic, oxalic,hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic,salicylic, nicotinic and 2-phenoxybenzoic.

Invention salts include those made from inorganic acids, e.g., HF, HCl,HBr, HI, H₂SO₄, H₃PO₄, Na₂CO₃, K₂CO₃, CaCO₃, MgCO₃ and NaClO₃. Suitableanions, which are optionally present with a cation such a Ca⁺⁺, Mg⁺⁺,Li⁺, Na⁺ or K⁺, include arsenate, arsenite formate, sorbate, chlorate,perchlorate, periodate, dichromate, glycodeoxycholate, cholate,deoxycholate, desoxycholate, taurocholate, taurodeoxycholate,taurolithocholate, tetraborate, nitrate, nitrite, sulfite, sulfamate,hyposulfite, bisulfite, metabisulfite, thiosulfate, thiocyanate,silicate, metasilicate, CN⁻, gluconate, gulcuronate, hippurate, picrate,hydrosulfite, hexafluorophosphate, hypochlorite, hypochlorate, borate,metaborate, tungstate and urate.

Salts also include the invention compound salts with one or more aminoacids. Many amino acids are suitable, especially the naturally-occurringamino acids found as protein components, although the amino acidtypically is one bearing a side chain with a basic or acidic group,e.g., lysine, arginine, histidine or glutamic acid, or a neutral groupsuch as glycine, serine, threonine, alanine, isoleucine, or leucine.

The invention compositions include compounds in their un-ionized, aswell as zwitterionic form, and combinations with stoiochimetric amountsof water as in hydrates.

Stereoisomers. The compounds of the invention (formula 1 compounds)include enriched or resolved optical isomers at any or all asymmetricatoms as are apparent from the depictions. Both racemic anddiasteromeric mixtures, as well as the individual optical isomers can beisolated or synthesized so as to be substantially free of theirenantiomeric or diastereomeric partners, and these are all within thescope of the invention. Chiral centers may be found in inventioncompounds at, for example, R¹, R², R³ and R⁴.

One or more of the following methods are used to prepare theenantiomerically enriched or pure isomers herein. The methods are listedin approximately their order of preference, i.e., one ordinarily shouldemploy stereospecific synthesis from chiral precursors beforechromatographic resolution before spontaneous crystallization.

Stereospecific synthesis is described in the examples. Methods of thistype conveniently are used when the appropriate chiral starting materialis available and reaction steps are chosen do not result in undesiredracemization at chiral sites. One advantage of stereospecific synthesisis that it does not produce undesired enantiomers that must be removedfrom the final product, thereby lowering overall synthetic yield. Ingeneral, those skilled in the art would understand what startingmaterials and reaction conditions should be used to obtain the desiredenantiomerically enriched or pure isomers by stereospecific synthesis.

If a suitable stereospecific synthesis cannot be empirically designed ordetermined with routine experimentation then those skilled in the artwould turn to other methods. One method of general utility ischromatographic resolution of enantiomers on chiral chromatographyresins. These resins are packed in columns, commonly called Pirklecolumns, and are commercially available. The columns contain a chiralstationary phase. The racemate is placed in solution and loaded onto thecolumn, and thereafter separated by HPLC. See for example, ProceedingsChromatographic Society-International Symposium on Chiral Separations,Sep. 3-4, 1987. Examples of chiral columns that could be used to screenfor the optimal separation technique would include Diacel Chriacel OD,Regis Pirkle Covalent D-phenylglycine, Regis Pirkle Type 1A, AstecCyclobond II, Astec Cyclobond III, Serva Chiral D-DL=Daltosil 100,Bakerbond DNBLeu, Sumipax OA-1000, Merck Cellulose Triacetate column,Astec Cyclobond I-Beta, or Regis Pirkle Covalent D-Naphthylalanine. Notall of these columns are likely to be effective with every racemicmixture. However, those skilled in the art understand that a certainamount of routine screening may be required to identify the mosteffective stationary phase. When using such columns it is desirable toemploy embodiments of the compounds of this invention in which thecharges are not neutralized, e.g., where acidic functionalities such ascarboxyl are not esterified or amidated.

Another method entails converting the enantiomers in the mixture todiasteriomers with chiral auxiliaries and then separating the conjugatesby ordinary column chromatography. This is a very suitable method,particularly when the embodiment contains free carboxyl, amino orhydroxyl that will form a salt or covalent bond to a chiral auxiliary.Chirally pure amino acids, organic acids or organosulfonic acids are allworthwhile exploring as chiral auxiliaries, all of which are well knownin the art. Salts with such auxiliaries can be formed, or they can becovalently (but reversibly) bonded to the functional group. For example,pure D or L amino acids can be used to amidate the carboxyl group ofinvention embodiments that comprise a carboxyl group and then separatedby chromatography.

Enzymatic resolution is another method of potential value. In suchmethods one prepares covalent derivatives of the enantiomers in theracemic mixture, generally lower alkyl esters (for example of carboxyl),and then exposes the derivative to enzymatic cleavage, generallyhydrolysis. For this method to be successful an enzyme must be chosenthat is capable of stereospecific cleavage, so it is frequentlynecessary to routinely screen several enzymes. If esters are to becleaved, then one selects a group of esterases, phosphatases, andlipases and determines their activity on the derivative. Typicalesterases are from liver, pancreas or other animal organs, and includeporcine liver esterase.

If the enatiomeric mixture separates from solution or a melt as aconglomerate, i.e., a mixture of enantiomerically pure crystals, thenthe crystals can be mechanically separated, thereby producing theenantiomerically enriched preparation. This method, however, is notpractical for large-scale preparations and is of limited value for trueracemic compounds.

Asymmetric synthesis is another technique for achieving enantiomericenrichment. For example, a chiral protecting group is reacted with thegroup to be protected and the reaction mixture allowed to equilibrate.If the reaction is enantiomerically specific then the product will beenriched in that enantiomer.

Further guidance in the separation of enantiomeric mixtures can befound, by way of example and not limitation, in “Enantiomers, Racemates,and resolutions”, Jean Jacques, Andre Collet, and Samuel H. Wilen(Krieger Publishing Company, Malabar, Fla., 1991, ISBN 0-89464-618-4):Part 2, Resolution of Enantiomer Mixture, pages 217-435; moreparticularly, section 4, Resolution by Direct Crystallization, pages217-251, section 5, Formation and Separation of Diastereomers, pages251-369, section 6, Crystallization-Induced Asymmetric Transformations,pages 369-378, and section 7, Experimental Aspects and Art ofResolutions, pages 378-435; still more particularly, section 5.1.4,Resolution of Alcohols, Transformation of Alcohols into Salt-FormingDerivatives, pages 263-266, section 5.2.3, Covalent Derivatives ofAlcohols, Thiols, and Phenols, pages 332-335, section 5.1.1, Resolutionof Acids, pages 257-259, section 5.1.2, Resolution of Bases, pages259-260, section 5.1.3, Resolution of Amino Acids, page 261-263, section5.2.1, Covalent Derivatives of Acids, page 329, section 5.2.2, Covalentderivatives of Amines, pages 330-331, section 5.2.4, CovalentDerivatives of Aldehydes, Ketones, and Sulfoxides, pages 335-339, andsection 5.2.7, Chromatographic Behavior of Covalent Diastereomers, pages348-354.

Unless otherwise stated or implied by context, expressions of apercentage of a liquid ingredient, e.g., an excipient, in an inventioncomposition or formulation mean the ingredient's percent by volume(v/v). Thus, 20% propylene glycol means 20% v/v propylene glycol ispresent in an invention composition or formulation. The amount ofexcipient indicated in invention compositions is not affected by theform used, e.g., NF or USP grade solvent or excipient. Thus, aninvention composition that comprises about 30% polyethylene glycol 300NF can instead comprise a USP counterpart, provided that otherlimitations, such as the amount of water present, are not exceeded.

As used herein, “innate immunity” refers to one or more componentstypically associated with nonspecific immune defense mechanisms in asubject. These components include the alternate complement pathway,e.g., Factor B, Factor D and properdin; NK cells, phagocytes (monocytes,macrophages), neutrophils, eosinophils, dendritic cells, fibrocytes;anti-microbial chemicals, e.g., defensins; physical barriers—skin,mucosal epithelium; and certain interleukins, chemokines and cytokines.Innate immunity plays a role in resistance to intracellular parasiteinfections, e.g., white blood cell infection, a liver infection, andother infections, e.g., lymph node infections. Enhancement of innateimmunity mechanism by formula 1 compounds or method described herein mayenhance phagolysosome fusion or movement, which some pathogens, e.g.,intracellular bacteria such as mycobacteria, or Listeria inhibit.

As used herein, references to CD molecules, specific immune cellsubsets, immune responses and the like, generally use nomenclature thatapplies to molecules, cells or the like that are found in humans.Analogs or counterparts of such molecules, cells or the like in otherspecies may have a differing nomenclature, but are included in thisinvention. A description of the nomenclature and function of various CDmolecules and immune cell subsets are as found in the scientificliterature. References to Th0, Th1 or Th2 cells and references to Th1 orTh2 immune responses in the context of human patients refers to thehuman counterparts of the murine Th0, Th1 or Th2 immune cells orresponses. For reviews see, e.g., A. K. Abbas et al., editors, Cellularand Molecular Immunology, W.B. Saunders Company, third edition, 1997,ISBN 0-7216-4024-9, pages 4-469, and I. Kimber and M. K. Selgrade,editors, T Lymphocyte Subpopulations in Immunotoxicology, John Wiley &Sons Ltd., 1998, ISBN 0-471-97194-4, pages 1-53.

“Immunosuppressive molecule” means molecules such as cyclosporin,cyclohexamide, mitomycin C, adriamycin, taxol and amphotericin B. Thesemolecules tend to have toxicities toward the immune system and aredirectly or indirectly immunosuppressive, i.e., toxic to dividing cellsor they can downregulate immunity.

“Steroid receptor” means a gene product, typically a protein monomer ordimer that can bind to a ligand, e.g., a natural steroid or an analogthereof, such as formula 1 compounds. Steroid receptors include orphansteroid receptors. Orphan steroid receptors are proteins for which thenatural ligand or biological function is at least partially unknown. Asused here, steroid receptors include homodimers, e.g., SXR and (CARβ)₂,and heterodimers, e.g., PXR-CARβ or RXR-CARβ. Steroid receptors alsoinclude isoforms, e.g., PXR.1 and PXR.2 for the PXR receptor, andhomologs of the steroid receptors, e.g., the homolog of CARβ known asMB67. Isoforms are typically generated by different splicing pathwaysfor a nuclear RNA from one gene, while homologs are typically a distinctcopy of a steroid receptor gene, where the gene copy encodes onlyrelatively small differences compared to the reference steroid receptorgene product. Such differences are most often found in areas other thanthe dimerization region and the steroid binding region of the steroidreceptor's structure. Typically isoforms and homologs bind the same orsimilar ligands as the reference gene product or steroid receptor.Steroid receptors may be of human or animal origin, e.g., obtained fromcells, tissues or cDNA expression libraries derived from cells ortissues of any primate, rodent (including murine), avian, ovine, bovine,equine, canine or feline species or any of the species or any specieswithin any group (e.g., Family or Genus) of species mentioned elsewhereherein or in any reference cited herein.

In the context of a combination of molecules that includes a steroidreceptor and a formula 1 compound, “invention complexes” or “complexes”means a complex that comprises a steroid receptor and a formula 1compound and optionally other molecules. These other molecules include(i) a DNA recognition sequence (“DNARS” hereafter), i.e., a sequencethat the steroid receptor specifically recognizes and binds to and (ii)a transcription factor that can bind to the steroid receptor-formula 1compound complex. As used herein, these complexes can arise in cells invitro or in vivo, or in cell-free systems. Complexes include, forexample, steroid receptor heterodimer-formula 1 compound combinations,steroid receptor homodimer-formula 1 compound combinations, steroidreceptor monomer-formula 1 compound combinations, steroid receptorheterodimer-formula 1 compound-DNA (or DNARS) combinations, steroidreceptor homodimer-formula 1 compound-DNA (or DNARS) combinations,steroid receptor heterodimer-formula 1 compound-transcription factorcombinations, steroid receptor homodimer-formula 1compound-transcription factor combinations, steroid receptorheterodimer-formula 1 compound-DNA (or DNARS)-transcription factorcombinations and steroid receptor homodimer-formula 1 compound-DNA (orDNARS)-transcription factor combinations.

An “agonist” or an “antagonist” is a compound or composition thatrespectively, either increases or decreases the activity of a receptor,which can lead to increased or decreased transcription of a regulatedgene. Receptors (and transcription factors) can modulate transcriptionof their target gene(s) by enhancing transcription or decreasing it.

General methods. Methods have been described, for example Karl Fischer(KF) and loss on drying (LOD), to determine the content of water orsolvents in various compositions. LOD measures all volatiles in asample, while KF is typically used to measure all water. When water isthe only volatile present, LOD values are equal to or less than KFvalues for a given composition. KF measures water in hydrates of acompound and LOD determines both water and the amount of other volatilesthat may be present. Invention compositions and formulations areconveniently assayed for water content by KF titration (e.g., using aMetrohm 684 KF Coulometer or equivalent) according to a publishedprocedure (U.S. Pharmacopoeia, vol. 23, 1995, chapter <921>, U.S.Pharmacopeial Convention, Inc., Rockville, Md.) and manufacturer'sCoulometer instructions. The amount of material used in the assay, about50-100 mg, is measured using a five place analytical balance (Sartorius,Model RC210D, or a suitable equivalent). The amounts of water specifiedin invention compositions and formulations is the amount obtained by KFanalysis.

Powder X-ray diffraction (XRD) methods have been used to characterizevarious crystalline compounds (see, e.g., U.S. Pharmacopoeia, volume 23,1995, <941>, p. 1843-1845, U.S. Pharmacopeial Convention, Inc.,Rockville, Md.; Stout et al., X-Ray Structure Determination; A PracticalGuide, MacMillan Co., New York, N.Y., 1986). The diffraction pattern, orportions thereof, obtained from a crystalline compound is usuallydiagnostic for a given crystal form, although weak or very weakdiffraction peaks may not always appear in replicate diffractionpatterns obtained from successive batches of crystals. Also, therelative intensities of XRD bands, particularly at low angle X-rayincidence values (low Theta), may vary due to preferred orientationeffects arising from differences in, e.g., crystal habit, particle sizeor other measurement conditions. Peaks on XRD spectra are typicallydefined at a given Theta value +/− about 0.1 to 0.2. XRD informationfrom the 1, 2, 3, 4, 5 or more main intensity XRD peaks, optionallycombined with one or more other diagnostic data (melting point, DSC,IR), is usually suitable to characterize or describe a crystallinematerial such as BrEA hemihydrate from other crystal forms that containthe same compound.

Other techniques that are used to identify or describe a crystallinematerial such as BrEA hemihydrate include melting point (MP),differential scanning calorimetry (DSC) and infrared absorptionspectroscopy (IR) data. DSC measures thermal transition temperatures atwhich a crystal absorbs or releases heat when its crystal structurechanges or it melts. MP data and DSC thermal transition temperatures aretypically reproducible within about 1 or 2° C. on successive analyses.IR measures absorption of infrared light that is associated with thepresence of particular chemical bonds that are associated with groups,e.g., hydroxyl, that vibrate in response to particular lightwavelengths.

Invention Embodiments. The invention provides BrEA hemihydrate, which istypically substantially free of other forms of BrEA, such as amorphousBrEA or anhydrous BrEA. As used herein, BrEA hemihydrate or crystallineBrEA hemihydrate refers to solid BrEA and water having an orderedarrangement of substantially all of the constituent molecules in adefined three-dimensional spatial pattern or lattice. Crystalline BrEAhemihydrate may comprise one or more crystal habits, e.g., tablets,rods, plates or needles. BrEA hemihydrate that is substantially free ofother forms of BrEA means a dry or substantially dry (where a liquid(s)comprises less than about 10% w/w of the total weight) solid preparationwhere more than about 55% w/w of the BrEA in the preparation is presentas BrEA hemihydrate. Such compositions typically comprise at least about60% w/w, or at least about 70% w/w, or at least about 80% w/w, usuallyat least about 90% w/w or at least about 95% w/w, or at least about 98%w/w of BrEA hemihydrate, with the remaining BrEA being present as otherforms of BrEA such as the amorphous or anhydrous BrEA. Solid BrEAhemihydrate will typically comprise at least about 90% w/w, usually atleast about 97% or about 98% w/w of the compound and less than about 10%w/w, usually less than about 3% or 2% w/w of by-products, which mayinclude the 16β isomer of BrEA or one or more by-products of BrEAsynthesis. Often the amount of solid BrEA that is present in a solid ora liquid medium will not contain detectable amounts of other forms ofBrEA (using standard analytical methods such as, e.g., FTIR, DSC or XRD)and the hemihydrate will may thus comprise about 99-100% w/w of thetotal amount of BrEA that is present.

Other invention embodiments include compositions that comprise asubstantial amount of BrEA hemihydrate that is present in compositionsthat comprise one or more other forms of BrEA, e.g., amorphous BrEA oranhydrous BrEA, and optionally one or more additional components, suchas any excipient described herein. As used herein, the “substantialamount” of BrEA hemihydrate in these compositions comprises at leastabout 15-20% w/w or at least about 20% w/w of BrEA hemihydrate of thetotal amount of BrEA that is present, typically at least about 25% w/w,more typically at least about 30% w/w, often at least about 35% w/w andusually at least about 45% w/w. These compositions are generally solids,e.g., formulations or unit dosages, but they also include suspensions,precipitates, gels and colloids that contain solid BrEA. Suchsuspensions or precipitates may arise from, e.g., precipitation of BrEAhemihydrate from a solution that contains water or from addition ofsolid BrEA to a liquid excipient(s). Obviously, compositions thatcomprise a substantial amount of BrEA may be substantially free of otherforms of solid BrEA as discussed above.

BrEA hemihydrate may conveniently be identified by reference to BrEAhemihydrate characterized by one or more of (1) its melting ordecomposition point or range (optionally expressed as +/−2° C.), (2) oneor more BrEA hemihydrate DSC transition temperatures or ranges (any ofwhich may be optionally expressed as +/−2° C.), (3) one or morecharacteristic BrEA hemihydrate IR absorption bands, (4) 1, 2, 3, 4, 5,6 or more of the highest intensity XRD peaks (any one or more of whichare optionally expressed as +/−0.1° Theta or +/−0.2° Theta) obtainedfrom an XRD spectrum of BrEA hemihydrate using Cu-Kα radiation (e.g.,obtained essentially according to the method described at U.S.Pharmacopoeia, volume 23,1995, <941>, p. 1843-1845), (5) the presence ofless than about 3% or less than about 2% w/w of other compounds, (6) awater content of dry BrEA hemihydrate of about 2.5% w/w (e.g., 2.3-2.7%w/w), where dry BrEA hemihydrate means compound dried by filtration,optionally washed once with an anhydrous solvent such as hexane,filtered again and dried in vacuo at about 60° C. until no furtherweight loss occurs over 24 hours at about 60° C. (e.g., where watercontent is determined essentially by the Karl Fisher or other methoddescribed at U.S. Pharmacopoeia, vol. 23,1995, p 1801-1802 or 1840-1843methods <731> or <921>), (7) cell constants and the orientation matrixobtained from single crystal X-ray crystallography of BrEA hemihydrate(obtained, e.g., essentially as described in WO 99/04774 at example 13),(8) a description of crystal shapes as observed at about 100×magnification to about 150× magnification by polarized light microscopyor (9) average BrEA hemihydrate crystal size and shape descriptions.

Thus, for example, BrEA hemihydrate may be characterized by or one ormore of its IR absorption bands, e.g., the carbonyl peaks at 1741 cm⁻¹and 1752 cm⁻¹, and either its melting or decomposition point or rangeand/or 1, 2, 3, 4, 5, 6 or more of the XRD peaks (usually the highestintensity peaks) at Theta (X-ray diffraction angle) values of 17.8,23.8, 24.2, 26.9-27.2, 28.6, 30.1 and 32.2.

BrEA hemihydrate is suitable to prepare compositions comprising anexcipient(s) suitable for human pharmaceutical use or for veterinaryuse. Such compositions are used to prepare formulations and unitdosages. Unit dosages typically comprise tablets, capsules, lozenges orsterile solutions, including sterile solutions for parenteraladministration. Solid unit dosage forms typically comprise about 5-1000mg of BrEA hemihydrate, typically about 20-400 mg, e.g., about 25 mg,about 50 mg, about 100 mg, about 150 mg or about 250 mg per unit dose.

The invention provides a method to make BrEA hemihydrate comprisingcontacting water, 16α-bromo-3β-hydroxy-5α-androstan-17-one and a C1-C6alcohol (e.g., methanol, ethanol, propanol, isopropanol, butanol) andwater. Typically the only one C1-C6 alcohol is present, e.g., ethanol,which is anhydrous or which may comprise up to about 2% w/w water. Insome embodiments, the method utilizes a solution that comprises about5-25% w/w water, about 30-45% w/w ethanol and about 30-45% w/w of a BrEApreparation. Typical BrEA preparations are solid preparations thatcomprise at least about 80% w/w, usually at least about 90% w/w or atleast about 95% w/w of BrEA. The solutions may comprise about 18-22% w/wwater, about 37-43% w/w ethanol and about 37-43% w/w of a BrEApreparation. In conducting the precipitation or crystallization method,the solution will typically be at a temperature of about −20° C. toabout 45° C., usually at about 0° C. to about 20° C. The solution ismaintained at this temperature range for about 30 minutes to about 12hours and the solution is optionally agitated using slow to moderateagitation during crystallization.

A related embodiment comprises a method to prepare BrEA hemihydratecomprising precipitating BrEA from a solution comprising at least about15-25% w/w water, about 35-45% w/w of a BrEA preparation and at leastabout 35-45% w/w of one or more water-miscible solvents, typically C₁₋₆alcohols (methanol, ethanol, propanol, isopropanol, butanol). The BrEApreparation may optionally comprise one or more by-products of BrEAsynthesis. Typical BrEA hemihydrate preparations or batches compriseless than about 5% w/w, usually less than about 3% or about 2% w/w ofother compounds, such as by-products of BrEA synthesis. Aspects of thismethod include contacting water with an organic solution that comprisesBrEA and an organic solvent such as a C1-C6 alcohol (e.g., ethanol) oracetone. Addition of water to such solutions leads to precipitation ofBrEA hemihydrate. Solutions that contain BrEA hemihydrate crystals orprecipitate are invention embodiments that are used to prepare solidBrEA that is later dried and stored, typically at ambient temperatures.

Precipitation of BrEA hemihydrate from water-containing solutions isaccomplished by known methods, e.g., reducing the solution'stemperature, using saturated or nearly saturated BrEA solutions, vacuumconcentration of saturated or nearly saturated BrEA solutions (which istypically conducted at a relatively low temperature, usually about15-25° C.), seeding with saturated or nearly saturated BrEA solutionswith BrEA hemihydrate crystals (e.g., about 10-100 mg per 1-10 L ofsolution), by heating a saturated or nearly saturated BrEA solution(about 25-35° C. for a few minutes followed by allowing the temperatureto fall or by actively cooling the solution) and optionally seeding thesolution with BrEA hemihydrate crystals or by addition of a liquid,e.g., additional water or ethanol, to a saturated or nearly saturatedBrEA ethanol-water solution, which causes the solution to becomesupersaturated. BrEA may also be precipitated from other solvents orsolvent systems, including acetone and acetone-ethanol. Such solventsare typically water miscible. Two-stage precipitation of BrEA may alsobe used to recover solid BrEA hemihydrate, e.g., initial precipitationand recovery of the solid, followed by either cooling and seeding of themother liquor or by allowing the mother liquor to stand, e.g., for aboutone, two or more days at ambient temperature, to obtain a second crop ofcrystals. Also, BrEA hemihydrate crystals may optionally berecrystallized, essentially as described herein, to further increase thepurity of the final solid. Methods for crystallizing organic compoundshave been described, e.g., A. S. Myerson, Handbook of IndustrialCrystallization, 1993, Butterworth-Heinemann, Stoneham, Mass., p 1-101.

Other related embodiments comprise a product produced by the process ofcontacting a solution comprising BrEA and an organic solvent with water.Typically the solutions are as described above, e.g., a solutioncomprising about 3-5% v/v water and at least about 40% v/v of one ormore water-miscible solvents, typically polar solvents such as C₁₋₆alcohols or ketones (e.g., methanol, ethanol, propanol, isopropanol,butanol, typically ethanol or acetone). Such processes are accomplishedby any one or more of the techniques described in the paragraph above,e.g., cooling of a saturated or nearly saturated BrEA water-ethanolsolution and optionally seeding the cooled solution with BrEAhemihydrate. An embodiment related to this comprises solutions or solidsthat comprise wet BrEA hemihydrate crystals or wet filtered orcentrifuged BrEA hemihydrate cakes, which may be obtained aftercrystallization. Examples of these embodiments include adding water to aBrEA-alcohol solution, e.g., slow addition of about 0.5-1.5 volumes orabout 0.8-1.2 volumes of water to about 6 volumes of a BrEA-ethanolsolution to obtain BrEA hemihydrate. Other examples of these embodimentsinclude adding water to a BrEA-ketone solvent solution, e.g., slowaddition of about 0.5-1.5 volumes or about 0.8-1.2 volumes of water toabout 10 volumes of a BrEA-acetone solution to obtain BrEA hemihydrate.

Another related embodiment is BrEA hemihydrate that is milled to anaverage particle size of about 0.01-200 μM, or about 0.1-10 μM or about0.5-5 μM. Average particle size or diameter for milled BrEA hemihydratemay thus be relatively small, e.g., about 0.03-2.0 μM or about 0.1-1.0μM, or somewhat larger, e.g., about 0.5-5.0 μM or about 1-5.0 μM. MilledBrEA hemihydrate is suitable for preparing solid formulations andparenteral formulations for human or veterinary use. The milled materialfacilitates dissolution of BrEA hemihydrate in solvents or excipientsand facilitates mixing with solids or solid excipients.

While it is possible to administer BrEA hemihydrate as a pure compoundto a subject, it is usually presented as a solid formulation or used toprepare a liquid formulation. Formulations will typically be used toprepare unit dosages, e.g., tablets, capsules or lozenges for oral,buccal or sublingual administration, that comprise about 10-1000 mg ortypically about 25-400 mg of BrEA hemihydrate. Alternatively,embodiments include a product for parenteral (e.g., subcutaneous,subdermal, intravenous, intramuscular, intraperitoneal) administrationmade by the process of contacting BrEA hemihydrate and a liquidexcipient, e.g., any one, two, three or more of PEG 100, PEG 200, PEG300, PEG 400, propylene glycol, benzyl benzoate, benzyl alcohol orethanol, and optionally sterilizing the solution and optionallydispensing the solution into vials or ampules (typically amber glass),which may be single-use or multi-use and optionally storing theformulation at reduced temperature (about 0-12° C., or about 2-10° C.).Such products for parenteral administration typically comprise BrEA at aconcentration of about 10-170 mg/mL, usually at about 20-110 mg/mL orabout 30-100 mg/mL, and optionally one or more of a salt, buffer orbacteriostat or preservative (e.g., NaCl, BHA, BHT or EDTA).

Other embodiments include a product produced by the process ofcontacting BrEA hemihydrate, which may be substantially free of otherforms of BrEA, with an excipient suitable for human pharmaceutical useor for veterinary use. The product is useful to make formulations orunit dosage forms that contain the hemihydrate.

Formulations made from or containing BrEA hemihydrate will usually bestored under conditions that limit the amount of water that reaches theformulation, e.g., silica gel in a sealed container that holds aformulation. Water permeation characteristics of containers have beendescribed, e.g., Containers-Permeation, Chapter, USP 23, 1995, U.S.Pharmacopeial Convention, Inc., Rockville, Md., p. 1787.

Embodiments include invention compositions that transiently occur when amethod step or operation is performed. For example, when a formula 1compound such as BrEA, containing less than about 3% water is contactedwith an excipient, e.g., a PEG, an alcohol, propylene glycol or benzylbenzoate, the composition before addition of one ingredient with anotheris a non-homogenous mixture. As the ingredients are contacted, themixture's homogeneity increases and the proportion of ingredientsrelative to each other approaches a desired value. Thus, inventioncompositions, which contain less than about 3% water can comprise about0.0001-99% of a formula 1 compound such as BrEA and one or moreexcipients. These transient compositions are intermediates thatnecessarily arise when one makes an invention composition or formulationand they are included in invention embodiments to the extent that theyare patentable.

In general, the formula 1 compound that is present in inventioncompositions and formulations is completely dissolved in the non-aqueousexcipients. However, in some embodiments, e.g., transient compositionsand some formulations, the formula 1 compound is partially dissolvedwhile the remaining portion is present as a solid, which can be asuspension or a colloid.

Invention compositions and formulations suitable for parenteral deliveryof formula 1 compounds to humans or animals typically comprise two,three or more excipients. Exemplary embodiments include (1) any two,three or four of propylene glycol, PEG200, PEG300, ethanol and benzylbenzoate and (2) any two, three or four of propylene glycol, PEG100,PEG200, PEG300, PEG400 and benzyl benzoate.

Invention compositions and formulations generally comprise about0.01-10% of BrEA, usually about 1-5%, and about 0.01-3% water, typicallyabout 0.05-3%, usually about 0.1-1%. The invention formulations areusually presented as unit or multi-unit dosages suitable for parenteraladministration once or twice per day or once per 2-3 days. Unit dosagescomprise about 3-1000 mg of BrEA per unit dose, typically about 5-500mg, usually about 10-200 mg. For treating retroviruses such as HIV inhumans, a unit dose usually comprises about 10-250 mg of BrEA, usuallyabout 100-200 mg, in a volume of about 1-6 mL, usually about 2-4 mL.

Invention embodiments include the product made by a process ofcombining, mixing or otherwise contacting BrEA and one, two or moreexcipients. Such products are produced by routine methods of contactingthe ingredients. Such products optionally also contain a diluent, adisintegrant and a binder, or other excipients described herein or inreferences cited herein.

BrEA in the presence of significant amounts of water was found toepimerize at the bromine atom, leading to a mixture of the 16α- and16β-BrEA isomers. Invention compositions and formulations comprisingBrEA or BrEA hemihydrate will usually have a water content of less thanabout 3%, typically less than about 0.3%, usually less than about 0.1%.These compositions and formulations were found to have a good stabilitywhen stored at ambient temperature (about 5-40° C. as used herein) inclosed containers compared to control compositions and formulationshaving more water. Such liquids are also characterized by an unexpectedreduction in precipitation of the compound, which water appears toinduce.

Invention embodiments include compositions that comprise less than about3% water, a formula 1 compound and a compound that is not generallyconsidered suitable for human use but is useful to make an inventionformulation for veterinary use. Veterinary formulations are compositionsuseful for the purpose of administering invention compositions toprimates, cats, dogs, horses, cows, rabbits and other subjects and maycontain excipients acceptable in the veterinary art and are compatiblewith formula 1 compounds such as BrEA. These veterinary compositions maynot always be suitable for human use because they contain an excipientthat is not suitable for human use, e.g., an alcohol other than ethanolsuch as methanol, propanol or butanol. Typically such excipients will bepresent at relatively low levels, e.g., about 1-30%, usually about 1-5%.

Invention embodiments include compositions and formulations, e.g., unitdosage forms and sterile solutions, that comprise (1) about 1-100 mg/mLof a formula 1 compound(s), about 57.5% propylene glycol, about 25%PEG300, about 12.5% ethanol and about 5% benzyl benzoate; (2) about 1-60mg/mL of a formula 1 compound(s), about 70% propylene glycol, about 25%PEG300 and about 5% benzyl benzoate; (3) about 1-60 mg/mL of a formula 1compound(s), about 25% PEG300, about 35% propylene glycol, about 35%mannitol and about 5% benzyl benzoate; (4) about 1-60 mg/mL of a formula1 compound(s), about 57.5% propylene glycol, a mixture comprising about25% PEG300 and PEG200 (e.g., PEG300:PEG200 in a ratio of about 1:10 toabout 10:1), about 12.5% ethanol and about 5% benzyl benzoate; (5) about1-60 mg/mL of a formula 1 compound(s), about 75% propylene glycol, amixture comprising about 25% PEG300 and PEG200 (e.g., a PEG300:PEG200 ina ratio of about 1:10 to about 10:1) and about 5% benzyl benzoate; (6)about 1-60 mg/mL of a formula 1 compound(s), about 25% PEG300 and PEG200(e.g., PEG300:PEG200 in a ratio of about 1:10 to about 10:1), about 35%propylene glycol, about 35% mannitol and about 5% benzyl benzoate; (7)any of formulations (1) through (6) where the formula 1 compound(s) isabout 40-55 mg/mL; (8) any of formulations (1) through (6) where theformula 1 compound(s) is about 30-100 mg/mL; (9) any of formulations (1)through (8) where 1, 2, 3 or 4 formula 1 compounds are present; (10) anyof formulations (1) through (8) where 1 or 2 formula 1 compounds arepresent; (11) any of formulations (1) through (8) where 1 formula 1compound is present; (12) any of formulations (1) through (11) where theformula 1 compound comprises independently at 1, 2 or 3 of any of R¹-R⁶,R¹⁰, R¹⁵, R¹⁷ or R¹⁸ an independently selected ester, thioester,carbonate, carbamate, amino acid or peptide of 1 or 2 independentlyselected formula 1 compounds; (13) any of formulations (1) through (12)where the formula 1 compound comprises or is BrEA or BrEA hemihydrate;(14) any of formulations (1) through (13) where the formula 1 compoundcomprises or is an ester, a sulfate ester or phosphoester of BrEA.

Other embodiments include the product obtained by storing inventioncompositions or formulations, e.g., unit dosage forms, any ofembodiments (1)-(14) above, or compositions used to make formulations,at about 10-40° C. for at least about 3 days, e.g., storage at ambienttemperature for about 1-24 months. Invention formulations will typicallybe stored in hermetically or induction sealed containers for these timeperiods. Invention compositions will typically be held in closedcontainers. The specification and claims disclose exemplary suitableformulations and unit dosage forms for these embodiments.

Other embodiments include compounds compositions and formulations whereone or more of R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸ comprise an amino acid or apeptide, e.g., R¹, R² or R⁴ comprises an amino acid or a peptide, R³ isa halogen and R⁵ and R⁶ are both —CH₃. The peptide at one or more ofR¹-⁶ can comprise a cell surface binding peptide such as the entireprotein or a sequence from fibronectin or retronectin, e.g., KQAGDV (SEQID NO 1).

In the formula 1 compounds, each R⁴ is independently selected. In someembodiments one R⁴ is hydrogen and the other is another moiety. In otherembodiments, both R⁴ are independently selected moieties other thanhydrogen, e.g., a C1 to C20 organic moiety.

R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸ include moieties, e.g., esters, thioesters,carbonates, amino acids, peptides and/or carbamates, that are chemicallyand/or enzymatically hydrolyzable, often under physiological conditions.Such moieties are independently chosen. Typically these moieties willgive rise to —OH, —SH or —NH₂ at the R¹-R⁶ positions of the steroidnucleus. Embodiments of formula 1 compounds include ones where (1) oneof R¹, R² and R⁴ is a hydrolyzable moiety (e.g., ester, thioester,carbonate, amino acid, peptide or carbamate), the other two of R¹, R²and R⁴ are —H, R³ is not hydrogen and R⁵ and R⁶ are both —CH₃, (2) twoof R¹, R² and R⁴ are hydrolyzable moieties (e.g., independently chosenesters, thioesters, carbonates, amino acids, peptides and/orcarbamates), the other of R¹, R² and R⁴ is —H, R³ is not hydrogen and R⁵and R⁶ are both —CH₃, (3) R¹, R² and R⁴ are hydrolyzable moieties, R³ isnot hydrogen and R⁵ and R⁶ are both —CH₃. In these embodiments, the R³group is typically in the β-configuration and the R¹, R² and R⁴-R⁶groups are typically in the α-configuration.

In other embodiments, one or more of R¹-R⁶, R¹⁰, R⁵, R¹⁷ and R¹⁸,usually one, comprises an amino acid or a peptide, while the remaininggroups are independently selected from the moieties defined herein. Inthese embodiments, the peptides are typically dimers (dipeptides) ortrimers (tripeptides). For example one of R¹, R² or R⁴ comprises anamino acid, the remaining of R¹, R² or R⁴ independently comprise —OH,═O, an ester, a carbonate or a carbamate, while R³ is a halogen,hydroxyl or an ester and R⁵ and R⁶ independently are —H, —(CH₂)_(n)—CH₃,—(CH₂)_(n)—CH₂OH, or —(CH₂)_(n)—CH₂F, —(CH₂)₂₋₄—O—(CH₂)₂₋₄—CH₃, where nis 0, 1, 2, 3, 4, 5, 6, 7 or 8 often 0, 1, or 2, usually 0. Typicallythe ester, carbonate or carbamate are hydrolyzable under physiologicalconditions.

Hydrolyzable moieties typically comprise acyl groups, esters, ethers,thioethers, amides, amino acids, peptides, carbonates and/or carbamates.In general, the structure of hydrolyzable moieties is not critical andcan vary. In some embodiments, these moieties contain a total of about 4to about 10 carbon atoms. These hydrolyzable moieties in otherembodiments comprise an organic moiety, as described above for ester,that contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms and 1,2, 3, 4, 5 or 6 heteroatoms, e.g., oxygen, nitrogen or sulfur. Thesehydrolyzable moieties can comprise no groups that are charged in plasma,blood, intracellular cytoplasm or in the gut, or they can comprise 1, 2,3 or more positive, negative or positive and negative charges under oneor more of these conditions. The charges may be fractional depending onthe group and the conditions it is under. These hydrolyzable moietiesmay comprise 1, 2, 3, 4 or more substitutions at a hydrogen atom(s)and/or a carbon atom(s), e.g., —OH, protected hydroxyl, —SH, protectedthiol, carboxyl, protected carboxyl, amine, protected amine, —O—, —S—,—CO—, —CS—, alkoxy, alkylthio, alkenyloxy, aryl, —OP(O)(O)—O—,—OS(O)(O)—O— and/or heterocycle. Such substitutions are independentlyselected.

Formula 1 compounds that comprise a hydrolyzable moiety(ies) may includeone or more independently chosen —O—CHR²⁴C(O)OR²⁵, —S—CHR²⁴C(O)OR²⁵,—NH—CHR²⁴C(O)OR²⁵, —O—CHR²⁴C(S)OR²⁵, —S—CHR²⁴C(S)OR²⁵,—NH—CHR²⁴C(S)OR²⁵, —O—CHR²⁴OC(O)R²⁵, —S—CHR²⁴OC(O)R²⁵,—NH—CHR²⁴OC(O)R²⁵, —O—CHR²⁴C(O)N(R²⁵)₂, —S—CHR²⁴C(O)N(R²⁵)₂,—NH—CHR²⁴C(O)N(R²⁵)₂, —O—CHR²⁴OR²⁵, —S—CHR²⁴OR²⁵, —NH—CHR²⁴OR²⁵,—O—CHR²⁴C(R²⁵)₂CH₂OX, —S—CHR²⁴C(R²⁵)₂CH₂OX, —NH—CHR²⁴C(R²⁵)₂CH₂OX,—O—CHR²⁴C(R²⁵)₂OX, —S—CHR²⁴C(R²⁵)₂OX or —NH—CHR²⁴C(R²⁵)₂OX, groups thatone or more of R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸ comprise these hydrolyzablemoieties, R²⁴ independently is —H, —CH₂—C₆H₅, —CH₂CH₂—C₆H₅, C₁₋₈ alkyl,C₂₋₈ alkenyl, aryl or heterocycle where each alkyl, alkenyl, aryl andheterocycle moiety is independently optionally substituted with 1, 2, or3, usually 1, —O—, —S—, —NH—, halogen, aryl, —OX, —SX, —NHX, ketone (═O)or —CN moieties or the C₁₋₈ alkyl is optionally substituted with 3, 4, 5or 6 halogens, and X is —H or a protecting group. Exemplary R²⁴ are —H,—CH₃, —C₂H₅, —CH₂—C₁₋₅ optionally substituted alkyl, —CH₂CH₂—C₁₋₄optionally substituted alkyl and —CH₂CH₂—O—C₁₋₄ optionally substitutedalkyl. R²⁵ independently is —H, —CH₂—C₆H₅, —CH₂CH₂—C₆H₅, C₁₋₁₂ alkyl,C₂₋₁₂ alkenyl, aryl, heterocycle, —CH₂-heterocycle or —CH₂-aryl, whereeach alkyl alkenyl, aryl, heterocycle, —CH₂-heterocycle or —CH₂-arylmoiety is independently optionally substituted with 1 or 2, usually 1,—O—, —S—, —NH—, halogen, aryl, —OX, —SX, —NHX, ketone (═O), —C(O)OX or—CN moieties or the C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl or aryl, are optionallyindependently substituted with 3, 4, 5 or 6 halogens, where X is —H or aprotecting group, or the aryl, heterocycle, —CH₂-heterocycle or—CH₂-aryl moieties are optionally independently substituted with 1, 2 or3 C₁₋₄ alkyl moieties or with 1, 2 or 3 C₁₋₄ alkoxy moieties at the arylmoiety or at the heterocycle, usually at a ring carbon. Exemplary R²⁵are —H, —CH₃, —C₂H₅, —C₃H₇, —C₄H₉, —C₆H₁₃, —C₆H₅, —C₆H₄OH, —C₆H₄OCH₃,—C₆H₄F, —CH₂—C₁₋₅ optionally substituted alkyl.

Embodiments of formula 1 compounds include or exclude any subset ofcompounds within the definition of formula 1, provided that at least onecompound remains. For example, a subset of formula 1 compounds that areusually included, for example in the invention nonaqueous formulationsand in the invention intermittent dosing protocols and immune modulationmethods, are formula 1 compounds where R² is hydroxyl, or a group thatcan hydrolyze to hydroxyl, in either configuration and R⁵ and R⁶ aremethyl in the α-configuration. A subset compounds that are optionallyexcluded from formula 1 compounds comprises one or all compounds thatare disclosed in one or more prior art references or publications, e.g.,one or more compounds that are disclosed in one or more of thereferences cited herein, especially for those compounds that can renderany claim or embodiment unpatentable for novelty, obviousness and/orinventive step reasons.

Exemplary embodiments of species and genera of formula 1 compounds arenamed as described below.

Group 1. Exemplary embodiments include the formula 1 compounds namedaccording to the compound structure designations given in Tables A and Bbelow. Each compound named in Table B is depicted as a compound havingformula B

where R⁵ and R⁶ are both —CH₃, there is no double bond at the 1-2-, 4-5-or 5-6-positions, one R⁴ is hydrogen, R⁷, R⁸ and R⁹ are all —CH₂— andR¹, R², R³ and R⁴ are the substituents designated in Table A. Thecompounds named according to Tables A and B are referred to as “group 1”compounds.

Compounds named in Table B are named by numbers assigned to R¹, R², R³and R⁴ according to the following compound naming convention, R¹, R²,R³, R⁴, based on the numbered chemical substituents depicted in Table A.Each Table A number specifies a different structure for each of R¹, R²,R³ and R⁴. When R¹, R², R³ or R⁴ is divalent moiety, e.g., ═O, thehydrogen at the corresponding position is absent. Thus, the group 1compound named 1.2.1.1 is a formula B structure with a β-hydroxyl bondedto carbons at the 3- and 7-positions (the variable groups R¹ and R²respectively), an α-bromine bonded to carbon 16 (the variable group R³)and double bonded oxygen (═O) at carbon 17 (the variable group R⁴),i.e., 1.2.1.1 has the structure shown below.

TABLE A R¹ 1 —OH 2 ═O 3 —SH 4 ═S 5 —O—CH₃ 6 —O—S(O)(O)—O⁻Na⁺ 7—O—S(O)(O)—OC₂H₅ 8 —CH₃ 9 —H 10 —OC(O)C(CH₃)₃ R² 1 —H 2 —OH 3 ═O 4 —CH₃5 —OCH₃ 6 —OC₂H₅ 7 —OCH₂CH₂CH₃ 8 —OCH₂CH₂CH₂CH₃ 9 —Cl 10 —Br R³ 1 —Br 2—Cl 3 —I 4 —F 5 —H 6 —OH 7 ═O 8 —O—C(O)—CH₃ 9 —O—C(O)—CH₂CH₃ 10—O—C(O)—CH₂CH₂CH₃ R⁴ 1 ═O 2 —OH 3 —H 4 —F 5 —Cl 6 —Br 7 —I 8 —O—C(O)—CH₃9 —O—C(O)—CH₂CH₃ 10 —O—C(O)—CH₂CH₂CH₃

TABLE B 1.1.1.1, 1.1.1.2, 1.1.1.3, 1.1.1.4, 1.1.1.5, 1.1.1.6, 1.1.1.7,1.1.1.8, 1.1.1.9, 1.1.1.10, 1.1.2.1, 1.1.2.2, 1.1.2.3, 1.1.2.4, 1.1.2.5,1.1.2.6, 1.1.2.7, 1.1.2.8, 1.1.2.9, 1.1.2.10, 1.1.3.1, 1.1.3.2, 1.1.3.3,1.1.3.4, 1.1.3.5, 1.1.3.6, 1.1.3.7, 1.1.3.8, 1.1.3.9, 1.1.3.10, 1.1.4.1,1.1.4.2, 1.1.4.3, 1.1.4.4, 1.1.4.5, 1.1.4.6, 1.1.4.7, 1.1.4.8, 1.1.4.9,1.1.4.10, 1.1.5.1, 1.1.5.2, 1.1.5.3, 1.1.5.4, 1.1.5.5, 1.1.5.6, 1.1.5.7,1.1.5.8, 1.1.5.9, 1.1.5.10, 1.1.6.1, 1.1.6.2, 1.1.6.3, 1.1.6.4, 1.1.6.5,1.1.6.6, 1.1.6.7, 1.1.6.8, 1.1.6.9, 1.1.6.10, 1.1.7.1, 1.1.7.2, 1.1.7.3,1.1.7.4, 1.1.7.5, 1.1.7.6, 1.1.7.7, 1.1.7.8, 1.1.7.9, 1.1.7.10, 1.1.8.1,1.1.8.2, 1.1.8.3, 1.1.8.4, 1.1.8.5, 1.1.8.6, 1.1.8.7, 1.1.8.8, 1.1.8.9,1.1.8.10, 1.1.9.1, 1.1.9.2, 1.1.9.3, 1.1.9.4, 1.1.9.5, 1.1.9.6, 1.1.9.7,1.1.9.8, 1.1.9.9, 1.1.9.10, 1.1.10.1, 1.1.10.2, 1.1.10.3, 1.1.10.4,1.1.10.5, 1.1.10.6, 1.1.10.7, 1.1.10.8, 1.1.10.9, 1.1.10.10, 1.2.1.1,1.2.1.2, 1.2.1.3, 1.2.1.4, 1.2.1.5, 1.2.1.6, 1.2.1.7, 1.2.1.8, 1.2.1.9,1.2.1.10, 1.2.2.1, 1.2.2.2, 1.2.2.3, 1.2.2.4, 1.2.2.5, 1.2.2.6, 1.2.2.7,1.2.2.8, 1.2.2.9, 1.2.2.10, 1.2.3.1, 1.2.3.2, 1.2.3.3, 1.2.3.4, 1.2.3.5,1.2.3.6, 1.2.3.7, 1.2.3.8, 1.2.3.9, 1.2.3.10, 1.2.4.1, 1.2.4.2, 1.2.4.3,1.2.4.4, 1.2.4.5, 1.2.4.6, 1.2.4.7, 1.2.4.8, 1.2.4.9, 1.2.4.10, 1.2.5.1,1.2.5.2, 1.2.5.3, 1.2.5.4, 1.2.5.5, 1.2.5.6, 1.2.5.7, 1.2.5.8, 1.2.5.9,1.2.5.10, 1.2.6.1, 1.2.6.2, 1.2.6.3, 1.2.6.4, 1.2.6.5, 1.2.6.6, 1.2.6.7,1.2.6.8, 1.2.6.9, 1.2.6.10, 1.2.7.1, 1.2.7.2, 1.2.7.3, 1.2.7.4, 1.2.7.5,1.2.7.6, 1.2.7.7, 1.2.7.8, 1.2.7.9, 1.2.7.10, 1.2.8.1, 1.2.8.2, 1.2.8.3,1.2.8.4, 1.2.8.5, 1.2.8.6, 1.2.8.7, 1.2.8.8, 1.2.8.9, 1.2.8.10, 1.2.9.1,1.2.9.2, 1.2.9.3, 1.2.9.4, 1.2.9.5, 1.2.9.6, 1.2.9.7, 1.2.9.8, 1.2.9.9,1.2.9.10, 1.2.10.1, 1.2.10.2, 1.2.10.3, 1.2.10.4, 1.2.10.5, 1.2.10.6,1.2.10.7, 1.2.10.8, 1.2.10.9, 1.2.10.10, 1.3.1.1, 1.3.1.2, 1.3.1.3,1.3.1.4, 1.3.1.5, 1.3.1.6, 1.3.1.7, 1.3.1.8, 1.3.1.9, 1.3.1.10, 1.3.2.1,1.3.2.2, 1.3.2.3, 1.3.2.4, 1.3.2.5, 1.3.2.6, 1.3.2.7, 1.3.2.8, 1.3.2.9,1.3.2.10, 1.3.3.1, 1.3.3.2, 1.3.3.3, 1.3.3.4, 1.3.3.5, 1.3.3.6, 1.3.3.7,1.3.3.8, 1.3.3.9, 1.3.3.10, 1.3.4.1, 1.3.4.2, 1.3.4.3, 1.3.4.4, 1.3.4.5,1.3.4.6, 1.3.4.7, 1.3.4.8, 1.3.4.9, 1.3.4.10, 1.3.5.1, 1.3.5.2, 1.3.5.3,1.3.5.4, 1.3.5.5, 1.3.5.6, 1.3.5.7, 1.3.5.8, 1.3.5.9, 1.3.5.10, 1.3.6.1,1.3.6.2, 1.3.6.3, 1.3.6.4, 1.3.6.5, 1.3.6.6, 1.3.6.7, 1.3.6.8, 1.3.6.9,1.3.6.10, 1.3.7.1, 1.3.7.2, 1.3.7.3, 1.3.7.4, 1.3.7.5, 1.3.7.6, 1.3.7.7,1.3.7.8, 1.3.7.9, 1.3.7.10, 1.3.8.1, 1.3.8.2, 1.3.8.3, 1.3.8.4, 1.3.8.5,1.3.8.6, 1.3.8.7, 1.3.8.8, 1.3.8.9, 1.3.8.10, 1.3.9.1, 1.3.9.2, 1.3.9.3,1.3.9.4, 1.3.9.5, 1.3.9.6, 1.3.9.7, 1.3.9.8, 1.3.9.9, 1.3.9.10,1.3.10.1, 1.3.10.2, 1.3.10.3, 1.3.10.4, 1.3.10.5, 1.3.10.6, 1.3.10.7,1.3.10.8, 1.3.10.9, 1.3.10.10, 1.4.1.1, 1.4.1.2, 1.4.1.3, 1.4.1.4,1.4.1.5, 1.4.1.6, 1.4.1.7, 1.4.1.8, 1.4.1.9, 1.4.1.10, 1.4.2.1, 1.4.2.2,1.4.2.3, 1.4.2.4, 1.4.2.5, 1.4.2.6, 1.4.2.7, 1.4.2.8, 1.4.2.9, 1.4.2.10,1.4.3.1, 1.4.3.2, 1.4.3.3, 1.4.3.4, 1.4.3.5, 1.4.3.6, 1.4.3.7, 1.4.3.8,1.4.3.9, 1.4.3.10, 1.4.4.1, 1.4.4.2, 1.4.4.3, 1.4.4.4, 1.4.4.5, 1.4.4.6,1.4.4.7, 1.4.4.8, 1.4.4.9, 1.4.4.10, 1.4.5.1, 1.4.5.2, 1.4.5.3, 1.4.5.4,1.4.5.5, 1.4.5.6, 1.4.5.7, 1.4.5.8, 1.4.5.9, 1.4.5.10, 1.4.6.1, 1.4.6.2,1.4.6.3, 1.4.6.4, 1.4.6.5, 1.4.6.6, 1.4.6.7, 1.4.6.8, 1.4.6.9, 1.4.6.10,1.4.7.1, 1.4.7.2, 1.4.7.3, 1.4.7.4, 1.4.7.5, 1.4.7.6, 1.4.7.7, 1.4.7.8,1.4.7.9, 1.4.7.10, 1.4.8.1, 1.4.8.2, 1.4.8.3, 1.4.8.4, 1.4.8.5, 1.4.8.6,1.4.8.7, 1.4.8.8, 1.4.8.9, 1.4.8.10, 1.4.9.1, 1.4.9.2, 1.4.9.3, 1.4.9.4,1.4.9.5, 1.4.9.6, 1.4.9.7, 1.4.9.8, 1.4.9.9, 1.4.9.10, 1.4.10.1,1.4.10.2, 1.4.10.3, 1.4.10.4, 1.4.10.5, 1.4.10.6, 1.4.10.7, 1.4.10.8,1.4.10.9, 1.4.10.10, 1.5.1.1, 1.5.1.2, 1.5.1.3, 1.5.1.4, 1.5.1.5,1.5.1.6, 1.5.1.7, 1.5.1.8, 1.5.1.9, 1.5.1.10, 1.5.2.1, 1.5.2.2, 1.5.2.3,1.5.2.4, 1.5.2.5, 1.5.2.6, 1.5.2.7, 1.5.2.8, 1.5.2.9, 1.5.2.10, 1.5.3.1,1.5.3.2, 1.5.3.3, 1.5.3.4, 1.5.3.5, 1.5.3.6, 1.5.3.7, 1.5.3.8, 1.5.3.9,1.5.3.10, 1.5.4.1, 1.5.4.2, 1.5.4.3, 1.5.4.4, 1.5.4.5, 1.5.4.6, 1.5.4.7,1.5.4.8, 1.5.4.9, 1.5.4.10, 1.5.5.1, 1.5.5.2, 1.5.5.3, 1.5.5.4, 1.5.5.5,1.5.5.6, 1.5.5.7, 1.5.5.8, 1.5.5.9, 1.5.5.10, 1.5.6.1, 1.5.6.2, 1.5.6.3,1.5.6.4, 1.5.6.5, 1.5.6.6, 1.5.6.7, 1.5.6.8, 1.5.6.9, 1.5.6.10, 1.5.7.1,1.5.7.2, 1.5.7.3, 1.5.7.4, 1.5.7.5, 1.5.7.6, 1.5.7.7, 1.5.7.8, 1.5.7.9,1.5.7.10, 1.5.8.1, 1.5.8.2, 1.5.8.3, 1.5.8.4, 1.5.8.5, 1.5.8.6, 1.5.8.7,1.5.8.8, 1.5.8.9, 1.5.8.10, 1.5.9.1, 1.5.9.2, 1.5.9.3, 1.5.9.4, 1.5.9.5,1.5.9.6, 1.5.9.7, 1.5.9.8, 1.5.9.9, 1.5.9.10, 1.5.10.1, 1.5.10.2,1.5.10.3, 1.5.10.4, 1.5.10.5, 1.5.10.6, 1.5.10.7, 1.5.10.8, 1.5.10.9,1.5.10.10, 1.6.1.1, 1.6.1.2, 1.6.1.3, 1.6.1.4, 1.6.1.5, 1.6.1.6,1.6.1.7, 1.6.1.8, 1.6.1.9, 1.6.1.10, 1.6.2.1, 1.6.2.2, 1.6.2.3, 1.6.2.4,1.6.2.5, 1.6.2.6, 1.6.2.7, 1.6.2.8, 1.6.2.9, 1.6.2.10, 1.6.3.1, 1.6.3.2,1.6.3.3, 1.6.3.4, 1.6.3.5, 1.6.3.6, 1.6.3.7, 1.6.3.8, 1.6.3.9, 1.6.3.10,1.6.4.1, 1.6.4.2, 1.6.4.3, 1.6.4.4, 1.6.4.5, 1.6.4.6, 1.6.4.7, 1.6.4.8,1.6.4.9, 1.6.4.10, 1.6.5.1, 1.6.5.2, 1.6.5.3, 1.6.5.4, 1.6.5.5, 1.6.5.6,1.6.5.7, 1.6.5.8, 1.6.5.9, 1.6.5.10, 1.6.6.1, 1.6.6.2, 1.6.6.3, 1.6.6.4,1.6.6.5, 1.6.6.6, 1.6.6.7, 1.6.6.8, 1.6.6.9, 1.6.6.10, 1.6.7.1, 1.6.7.2,1.6.7.3, 1.6.7.4, 1.6.7.5, 1.6.7.6, 1.6.7.7, 1.6.7.8, 1.6.7.9, 1.6.7.10,1.6.8.1, 1.6.8.2, 1.6.8.3, 1.6.8.4, 1.6.8.5, 1.6.8.6, 1.6.8.7, 1.6.8.8,1.6.8.9, 1.6.8.10, 1.6.9.1, 1.6.9.2, 1.6.9.3, 1.6.9.4, 1.6.9.5, 1.6.9.6,1.6.9.7, 1.6.9.8, 1.6.9.9, 1.6.9.10, 1.6.10.1, 1.6.10.2, 1.6.10.3,1.6.10.4, 1.6.10.5, 1.6.10.6, 1.6.10.7, 1.6.10.8, 1.6.10.9, 1.6.10.10,1.7.1.1, 1.7.1.2, 1.7.1.3, 1.7.1.4, 1.7.1.5, 1.7.1.6, 1.7.1.7, 1.7.1.8,1.7.1.9, 1.7.1.10, 1.7.2.1, 1.7.2.2, 1.7.2.3, 1.7.2.4, 1.7.2.5, 1.7.2.6,1.7.2.7, 1.7.2.8, 1.7.2.9, 1.7.2.10, 1.7.3.1, 1.7.3.2, 1.7.3.3, 1.7.3.4,1.7.3.5, 1.7.3.6, 1.7.3.7, 1.7.3.8, 1.7.3.9, 1.7.3.10, 1.7.4.1, 1.7.4.2,1.7.4.3, 1.7.4.4, 1.7.4.5, 1.7.4.6, 1.7.4.7, 1.7.4.8, 1.7.4.9, 1.7.4.10,1.7.5.1, 1.7.5.2, 1.7.5.3, 1.7.5.4, 1.7.5.5, 1.7.5.6, 1.7.5.7, 1.7.5.8,1.7.5.9, 1.7.5.10, 1.7.6.1, 1.7.6.2, 1.7.6.3, 1.7.6.4, 1.7.6.5, 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10.7.1.2, 10.7.1.3, 10.7.1.4, 10.7.1.5,10.7.1.6, 10.7.1.7, 10.7.1.8, 10.7.1.9, 10.7.1.10, 10.7.2.1, 10.7.2.2,10.7.2.3, 10.7.2.4, 10.7.2.5, 10.7.2.6, 10.7.2.7, 10.7.2.8, 10.7.2.9,10.7.2.10, 10.7.3.1, 10.7.3.2, 10.7.3.3, 10.7.3.4, 10.7.3.5, 10.7.3.6,10.7.3.7, 10.7.3.8, 10.7.3.9, 10.7.3.10, 10.7.4.1, 10.7.4.2, 10.7.4.3,10.7.4.4, 10.7.4.5, 10.7.4.6, 10.7.4.7, 10.7.4.8, 10.7.4.9, 10.7.4.10,10.7.5.1, 10.7.5.2, 10.7.5.3, 10.7.5.4, 10.7.5.5, 10.7.5.6, 10.7.5.7,10.7.5.8, 10.7.5.9, 10.7.5.10, 10.7.6.1, 10.7.6.2, 10.7.6.3, 10.7.6.4,10.7.6.5, 10.7.6.6, 10.7.6.7, 10.7.6.8, 10.7.6.9, 10.7.6.10, 10.7.7.1,10.7.7.2, 10.7.7.3, 10.7.7.4, 10.7.7.5, 10.7.7.6, 10.7.7.7, 10.7.7.8,10.7.7.9, 10.7.7.10, 10.7.8.1, 10.7.8.2, 10.7.8.3, 10.7.8.4, 10.7.8.5,10.7.8.6, 10.7.8.7, 10.7.8.8, 10.7.8.9, 10.7.8.10, 10.7.9.1, 10.7.9.2,10.7.9.3, 10.7.9.4, 10.7.9.5, 10.7.9.6, 10.7.9.7, 10.7.9.8, 10.7.9.9,10.7.9.10, 10.7.10.1, 10.7.10.2, 10.7.10.3, 10.7.10.4, 10.7.10.5,10.7.10.6, 10.7.10.7, 10.7.10.8, 10.7.10.9, 10.7.10.10, 10.8.1.1,10.8.1.2, 10.8.1.3, 10.8.1.4, 10.8.1.5, 10.8.1.6, 10.8.1.7, 10.8.1.8,10.8.1.9, 10.8.1.10, 10.8.2.1, 10.8.2.2, 10.8.2.3, 10.8.2.4, 10.8.2.5,10.8.2.6, 10.8.2.7, 10.8.2.8, 10.8.2.9, 10.8.2.10, 10.8.3.1, 10.8.3.2,10.8.3.3, 10.8.3.4, 10.8.3.5, 10.8.3.6, 10.8.3.7, 10.8.3.8, 10.8.3.9,10.8.3.10, 10.8.4.1, 10.8.4.2, 10.8.4.3, 10.8.4.4, 10.8.4.5, 10.8.4.6,10.8.4.7, 10.8.4.8, 10.8.4.9, 10.8.4.10, 10.8.5.1, 10.8.5.2, 10.8.5.3,10.8.5.4, 10.8.5.5, 10.8.5.6, 10.8.5.7, 10.8.5.8, 10.8.5.9, 10.8.5.10,10.8.6.1, 10.8.6.2, 10.8.6.3, 10.8.6.4, 10.8.6.5, 10.8.6.6, 10.8.6.7,10.8.6.8, 10.8.6.9, 10.8.6.10, 10.8.7.1, 10.8.7.2, 10.8.7.3, 10.8.7.4,10.8.7.5, 10.8.7.6, 10.8.7.7, 10.8.7.8, 10.8.7.9, 10.8.7.10, 10.8.8.1,10.8.8.2, 10.8.8.3, 10.8.8.4, 10.8.8.5, 10.8.8.6, 10.8.8.7, 10.8.8.8,10.8.8.9, 10.8.8.10, 10.8.9.1, 10.8.9.2, 10.8.9.3, 10.8.9.4, 10.8.9.5,10.8.9.6, 10.8.9.7, 10.8.9.8, 10.8.9.9, 10.8.9.10, 10.8.10.1, 10.8.10.2,10.8.10.3, 10.8.10.4, 10.8.10.5, 10.8.10.6, 10.8.10.7, 10.8.10.8,10.8.10.9, 10.8.10.10, 10.9.1.1, 10.9.1.2, 10.9.1.3, 10.9.1.4, 10.9.1.5,10.9.1.6, 10.9.1.7, 10.9.1.8, 10.9.1.9, 10.9.1.10, 10.9.2.1, 10.9.2.2,10.9.2.3, 10.9.2.4, 10.9.2.5, 10.9.2.6, 10.9.2.7, 10.9.2.8, 10.9.2.9,10.9.2.10, 10.9.3.1, 10.9.3.2, 10.9.3.3, 10.9.3.4, 10.9.3.5, 10.9.3.6,10.9.3.7, 10.9.3.8, 10.9.3.9, 10.9.3.10, 10.9.4.1, 10.9.4.2, 10.9.4.3,10.9.4.4, 10.9.4.5, 10.9.4.6, 10.9.4.7, 10.9.4.8, 10.9.4.9, 10.9.4.10,10.9.5.1, 10.9.5.2, 10.9.5.3, 10.9.5.4, 10.9.5.5, 10.9.5.6, 10.9.5.7,10.9.5.8, 10.9.5.9, 10.9.5.10, 10.9.6.1, 10.9.6.2, 10.9.6.3, 10.9.6.4,10.9.6.5, 10.9.6.6, 10.9.6.7, 10.9.6.8, 10.9.6.9, 10.9.6.10, 10.9.7.1,10.9.7.2, 10.9.7.3, 10.9.7.4, 10.9.7.5, 10.9.7.6, 10.9.7.7, 10.9.7.8,10.9.7.9, 10.9.7.10, 10.9.8.1, 10.9.8.2, 10.9.8.3, 10.9.8.4, 10.9.8.5,10.9.8.6, 10.9.8.7, 10.9.8.8, 10.9.8.9, 10.9.8.10, 10.9.9.1, 10.9.9.2,10.9.9.3, 10.9.9.4, 10.9.9.5, 10.9.9.6, 10.9.9.7, 10.9.9.8, 10.9.9.9,10.9.9.10, 10.9.10.1, 10.9.10.2, 10.9.10.3, 10.9.10.4, 10.9.10.5,10.9.10.6, 10.9.10.7, 10.9.10.8, 10.9.10.9, 10.9.10.10, 10.10.1.1,10.10.1.2, 10.10.1.3, 10.10.1.4, 10.10.1.5, 10.10.1.6, 10.10.1.7,10.10.1.8, 10.10.1.9, 10.10.1.10, 10.10.2.1, 10.10.2.2, 10.10.2.3,10.10.2.4, 10.10.2.5, 10.10.2.6, 10.10.2.7, 10.10.2.8, 10.10.2.9,10.10.2.10, 10.10.3.1, 10.10.3.2, 10.10.3.3, 10.10.3.4, 10.10.3.5,10.10.3.6, 10.10.3.7, 10.10.3.8, 10.10.3.9, 10.10.3.10, 10.10.4.1,10.10.4.2, 10.10.4.3, 10.10.4.4, 10.10.4.5, 10.10.4.6, 10.10.4.7,10.10.4.8, 10.10.4.9, 10.10.4.10, 10.10.5.1, 10.10.5.2, 10.10.5.3,10.10.5.4, 10.10.5.5, 10.10.5.6, 10.10.5.7, 10.10.5.8, 10.10.5.9,10.10.5.10, 10.10.6.1, 10.10.6.2, 10.10.6.3, 10.10.6.4, 10.10.6.5,10.10.6.6, 10.10.6.7, 10.10.6.8, 10.10.6.9, 10.10.6.10, 10.10.7.1,10.10.7.2, 10.10.7.3, 10.10.7.4, 10.10.7.5, 10.10.7.6, 10.10.7.7,10.10.7.8, 10.10.7.9, 10.10.7.10, 10.10.8.1, 10.10.8.2, 10.10.8.3,10.10.8.4, 10.10.8.5, 10.10.8.6, 10.10.8.7, 10.10.8.8, 10.10.8.9,10.10.8.10, 10.10.9.1, 10.10.9.2, 10.10.9.3, 10.10.9.4, 10.10.9.5,10.10.9.6, 10.10.9.7, 10.10.9.8, 10.10.9.9, 10.10.9.10, 10.10.10.1,10.10.10.2, 10.10.10.3, 10.10.10.4, 10.10.10.5, 10.10.10.6, 10.10.10.7,10.10.10.8, 10.10.10.9, 10.10.10.10

Additional exemplary formula B compound groups include the followingcompound groups disclosed below. Unless otherwise specified, theconfigurations of all hydrogen atoms and R groups for the followingcompound groups are as defined for the group 1 compounds of formula Babove.

Group 2. This group comprises compounds named in Table B having R¹, R²,R³ and R⁴ substituents defined in Table A wherein the R¹, R², R³ and R⁴substituents are bonded to the steroid nucleus described for group 1compounds, except that a double bond at the 5-6 position is present.Thus, group 2 compound 1.3.1.1 has the structure

Group 3. This group comprises compounds named in Table B having R¹, R²,R³ and R⁴ substituents defined in Table A wherein the R¹, R², R³ and R⁴substituents are bonded to the steroid nucleus as described for group 1compounds, except that double bonds at the 1-2- and 5-6 positions arepresent. Thus, group 3 compound 2.2.5.1 has the structure

Group 4. This group comprises compounds named in Table B having R¹, R²,R³ and R⁴ substituents defined in Table A wherein the R¹, R², R³ and R⁴substituents are bonded to the steroid nucleus described for group 1compounds, except that a double bond at the 1-2 position is present.Thus, group 4 compound 5.2.7.8 has the structure

Group 5. This group comprises compounds named in Table B having R¹, R²,R³ and R⁴ substituents defined in Table A wherein the R¹, R², R³ and R⁴substituents are bonded to the steroid nucleus described for group 1compounds, except that a double bond at the 4-5 position is present.Thus, the group 5 compound named 3.5.2.9 has the structure

Group 6. This group comprises compounds named in Table B having R¹, R²,R³ and R⁴ substituents defined in Table A wherein the R¹, R², R³ and R⁴substituents are bonded to the steroid nucleus described for group 1compounds, except that double bonds at both the 1-2 and 4-5 positionsare present. Thus, the group 6 compound named 10.2.7.8 has the structure

Groups 7-1 through 7-6. These groups comprise the 6 compound groupsdescribed above, except that R⁵ is hydrogen instead of methyl. Thus,group 7-1 has the same steroid nucleus as group 1 above, i.e., no doublebond is present, but R⁵ is —H. Group 7-2 comprises the same steroidnucleus as group 2 above, i.e., a double bond is present at the5-6-position, but R⁵ is —H, Compound groups 7-3 through 7-6 are assigneda steroid nucleus in the same manner. Thus, the group 7-1 through group7-6 compounds named 1.2.1.9 have the structures

Groups 8-1 through 8-6. These groups comprise each compound named ingroups 1-6, except that R⁵ of formula B is —CH₂OH instead of methyl. Thegroups 8-1 through group 8-6 compounds have structures that are named inthe same manner as group 1-6 compounds, except that —CH₂OH instead ofmethyl is present at R⁵. These groups are named in the same manner asgroups 7-1 through 7-6. Thus, group 8-1 and group 8-2 compounds named1.2.1.9 have the structures

Groups 9-1 through 9-6. These groups comprise each compound named incompound groups 1-6, except that R⁶ of formula B is hydrogen instead ofmethyl. The groups 9-1 through group 9-6 compounds have structures thatare named in the same manner as group 7-1 through 7-6 compounds, exceptthat —H instead of methyl is present at R⁶. Thus, group 9-1 and group9-2 compounds named 1.2.1.9 have the structures

Groups 10-1 through 10-6. These groups comprise each compound named incompound groups 1-6 where R⁶ of formula 1 is —CH₂OH instead of methyl.The groups 10-1 through group 10-6 compounds have structures that arenamed in the same manner as group 7-1 through 7-6 compounds, except that—CH₂OH instead of methyl is present at R⁶. Thus, group 10-1 and group10-2 compounds named 1.2.1.9 have the structures

Groups 11-1 through 11-10-6. These groups comprise each compound namedin compound groups 1 through 10-6 where R¹ substituents 1-10 listed inTable A are replaced with the following substituents:

-   1 —O—C(O)—CH₂CH₂CH₂CH₃ (—O—C(O)—CH₂CH₂CH₂CH₃ replaces —OH, which is    R¹ substituent 1 in Table A)-   2 —O—C(O)—CH₂CH₂CH₂CH₂CH₂CH₃-   3 —O—C(O)—CH₂CH₂OCH₂CH₃-   4 —O—C(O)—CH₂CH₂OCH₂CH₂OCH₂CH₃-   5 —O—C(O)—CH₂CH₂CH₂CH₂OCH₂CH₃-   6 —O—C(O)—CH₂CH₂OCH₂CH₂CH₂CH₃-   7 —O—C₆H₄Cl-   8 —O—C₆H₃F₂-   9 —O—C₆H₄—O(CH₂)₂—O—CH₂CH₃-   10 —O—C₆H₄—C(O)O(CH₂)₀₋₉CH₃

The group 11-1 through group 11-6 compounds have structures that arenamed in the same manner as group 7-1 through 7-6 compounds, except thatsubstituents 1-10 of table A are replaced by the substituents 1-10 at R¹listed above. Thus group 11-1 and 11-2 compounds named 1.2.1.9 have thestructures

Group 11-7-1 and 11-7-2 compounds named 1.2.1.9 have the structures

Group 11-8-1 and 11-8-2 compounds named 1.2.1.9 have the structures

Groups 12-1 through 12-10-6. These groups comprise each compound namedin groups 1 through 10-6 where R¹ substituents 1-10 listed in Table Aare replaced with the following groups:

-   1 —O—P(O)(O)—OCH₂CH(CH₃)CH₃ (—O—P(O)(O)—OCH₂CH(CH₃)CH₃ replaces —OH,    which is R¹ substituent 1 in Table A)-   2 —O—P(O)(O)—OCH₂CH₂CH₂CH₂CH₃-   3 —O—P(O)(O)—OCH₂CH₂CH₂CH₂CH₂CH₃-   4 —O—P(O)(O)—OCH₂CH₂CH(CH₂CH₂)CH₃-   5 —O—CH₂CH₂CH₂CH₂CH₂CH₃-   6 —O—C₂H₅-   7 —O—CH₂CH₂CH₃-   8 —O—CH₂CH₂CH₂CH₃-   9 —O—CH(CH₃)CHCH₃-   10 —O—C(CH₃)₃

Groups 13-1 through 13-10-6. These groups comprise each compound namedin groups 1 through 10-6 where R¹ substituents 1-10 listed in Table Aare replaced with the following groups:

-   1 —O—(CH₂)₄CH₃ (—O—(CH₂)₄CH₃ replaces —OH, which is R¹ substituent 1    in Table A)-   2 —O—C(O)—NH₂-   3 —O—C(O)—NHCH₃-   4 —O—C(O)—NHC₂H₅-   5 —O—C(O)—NHCH₂CH₂CH₃-   6 —O—C(O)—NHCH₂CH₂OCH₂CH₃-   7 —O—C(O)—CH₃-   8 —O—C(O)—C₂H₅-   9 —O—C(O)—CH₂CH₂CH₃-   10 —O—C(O)—CH₂CH₂CH₂CH₃

Groups 14-1 through 14-10-6. These groups comprise each compound namedin groups 1 through 10-6 where R¹ substituents 1-10 listed in Table Aare replaced with the following groups:

-   1 —O—CH₂C₆H₅-   2 —O—CH₂C₆H₅-   3 —O—CH₂C₆H₄OCH₃-   4 —O—CH₂C₆H₄OCH₃-   5 —O—CH₂C₆H₄F-   6 —O—CH₂C₆H₄F-   7 —O—CH₂C₆H₃(OCH₃)₂-   8 —O—CH₂C₆H₃(OCH₃)₂-   9 —O—CH₂C₆H₄OCH₂CH₃-   10 —O—CH₂C₆H₄OCH₂CH₃

Groups 15-1 through 15-10-6. These groups comprise each compound namedin groups 1 through 10-6 where R¹ substituents 1-10 listed in Table Aare replaced with the following groups:

-   1 —O—C(O)—CH₂CH₂NH₂ (—O—C(O)—CH₂CH₂NH₂ replaces —OH, which is R¹    substituent 1 in Table A)-   2 —O—C(O)—CH₂CH₂CH₂NH₂-   3 —O—C(O)—CH₂OH-   4 —O—C(O)—CH₂CH₂OH-   5 —O—C(O)—CH₂CH₂CH₂OH-   6 —O—C(O)—CH₂SH-   7 —O—C(O)—CH₂CH₂SH-   8 —O—C(O)—CH₂CH₂CH₂SH-   9 —O—S(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—C₂H₅-   10 —O—P(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—C₂H₅

Groups 16-1 through 16-10-6. These groups comprise each compound namedin groups 1 through 10-6 where R¹ substituents 1-10 listed in Table Aare replaced with the following groups:

-   1 —O—C(O)-A4-NH₂, where A4-NH₂ is a 4 carbon alkyl group substituted    with —NH₂ (—O—C(O)-A4-NH₂ replaces —OH, which is R¹ substituent 1 in    Table A)-   2 —O—C(O)-A6-NH₂, where A6-NH₂ is a 6 carbon alkyl group substituted    with —NH₂-   3 —O—C(O)-A8-NH₂, where A8-NH₂ is a 8 carbon alkyl group substituted    with —NH₂-   4 —O—C(O)-A4-OH, where A4-OH is a 4 carbon alkyl group substituted    with —OH or —O—-   5 —O—C(O)-A6-OH, where A6-OH is a 6 carbon alkyl group substituted    with —OH or —O—-   6—O—C(O)-A8-OH, where A8-OH is a 8 carbon alkyl group substituted    with —OH or —O—-   7 —O—S(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—C₃H₇-   8 —O—P(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—C₃H₇-   9 —O—S(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—C₄H₉-   10 —O—P(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—C₄H₉

Groups 17-1 through 17-10-6. These groups comprise each compound namedin compound groups 1 through 10-6 where R¹ substituents 1-10 listed inTable A are replaced with the following groups:

-   1 —O—S(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—C₆H₁₃-   2 —O—P(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—C₆H₁₃-   3 —O—S(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—C₈H₁₇-   4 —O—P(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—C₈H₁₇-   5 —O—S(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—CH₂C₅H₁₀OH-   6 —O—P(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—CH₂C₅H₁₀OH-   7 —O—S(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—CH₂C₃H₆OH-   8 —O—P(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—CH₂C₃H₆OH-   9 —O—S(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—CH₂C₇H₁₄OH-   10 —O—P(O)(O)—O—CH₂—CH(O—C(O)—OH)—CH₂—O—C(O)—CH₂C₇H₁₄OH

Groups 18-1 through 18-10-6. These groups comprise each compound namedin groups 1 through 10-6 where R⁴ substituents 1-10 listed in Table Aare replaced with the following groups:

-   1 —O—C(O)CH₂NH₂-   2 —O—C(O)C(CH₃)H—NH₂-   3 —O—C(O)C(CH₂C₆H₅)H—NH₂-   4 —O—C(O)—O—NHC(CH₃)H—CO₂H-   5 —O—C(O)—O—NHCH₂—CO₂H-   6 —O—C(O)—O—NH(CH₂C₆H₅)H—CO₂H-   7 —O—C(O)—CF₃-   8 —O—C(O)—CH₂CF₃-   9 —O—C(O)—(CH₂)₃CF₃-   10 —O—C(O)—(CH₂)₅CH₃

Groups 19-1 through 19-10-6. These groups comprise each compound namedin compound groups 1 through 10-6 where R⁴ substituents 1-10 listed inTable A are replaced with the following groups:

-   1 —O—C(O)—O—CH₃-   2 —O—C(O)—O—CH₂CH₃-   3 —O—C(O)—O—C₃H₇-   4 —O—C(O)—O—C₄H₉-   5 —O—C(O)—O—C₆H₁₃-   6 —O—C(O)—O—C₆H₅-   7 —O—C(O)—O—C₆H₄OH-   8 —O—C(O)—O—C₆H₄OCH₃-   9 —O—C(O)—O—C₆H₄OCH₂CH₃-   10 —O—C(O)—O—C₆H₄F

Groups 20-1 through 20-10-6. These groups comprise each compound namedin groups 1 through 10-6 where R⁴ substituents 1-10 listed in Table Aare replaced with the following groups:

-   1 —O—C(O)—S—CH₃-   2 —O—C(O)—S—CH₂CH₃-   3 —O—C(O)—S—C₃H₇-   4 —O—C(O)—S—C₄H₉-   5 —O—C(O)—S—C₆H₁₃-   6 —O—C(O)—S—C₆H₅-   7 —O—C(O)—S—C₆H₄OH-   8 —O—C(O)—S—C₆H₄OCH₃-   9 —O—C(O)—S—C₆H₄OCH₂CH₃-   10 —O—C(O)—S—C₆H₄F

Groups 21-1 through 21-10-6. These groups comprise each compound namedin compound groups 1 through 10-6 where R⁴ substituents 1-10 listed inTable A are replaced with the following groups:

-   1 —O—C(S)—O—CH₃-   2 —O—C(S)—O—CH₂CH₃-   3 —SH-   4 ═S-   5 —O—C(S)—O—C₆H₁₃-   6 —O—C(O)—O—CH₂C₆H₅-   7 —O—C(O)—O—CH₂C₆H₄OH-   8 —O—C(O)—O—CH₂C₆H₄OCH₃-   9 —O—C(O)—O—CH₂C₆H₄OCH₂CH₃-   10 —O—C(O)—O—CH₂C₆H₄F

Groups 22-1 through 22-10-6. These groups comprise each compound namedin compound groups 1 through 10-6 where R² substituents 1-10 listed inTable A are replaced with the following groups:

-   1 —O—C(S)—O—CH₃-   2 —O—C(S)—O—CH₂CH₃-   3 —O—C(S)—O—C₃H₇-   4 —O—C(S)—O—C₄H₉-   5 —O—C(S)—O—C₆H₁₃-   6 —O—C(O)—O—CH₂C₆H₅-   7 —O—C(O)—O—CH₂C₆H₄OH-   8 —O—C(O)—O—CH₂C₆H₄OCH₃-   9 —O—C(O)—O—CH₂C₆H₄OCH₂CH₃-   10 —O—C(O)—O—CH₂C₆H₄F

Groups 23-1 through 23-10-6. These groups comprise each compound namedin compound groups 1 through 10-6 where R³ substituents 1-10 listed inTable A are replaced with the following groups:

-   1 —O—C(S)—O—CH₃-   2 —O—C(S)—O—CH₂CH₃-   3 —O—C(S)—O—C₃H₇-   4 —O—C(S)—O—C₄H₉-   5 —O—C(S)—O—C₆H₁₃-   6 —O—C(O)—O—CH₂C₆H₅-   7 —O—C(O)—O—CH₂C₆H₄OH-   8 —O—C(O)—O—CH₂C₆H₄OCH₃-   9 —O—C(O)—O—CH₂C₆H₄OCH₂CH₃-   10 —O—C(O)—O—CH₂C₆H₄F

Groups 24-1 through 24-10-6. These groups comprise each compound namedin compound groups 1 through 10-6 where R² substituents 1-10 listed inTable A are replaced with the following groups:

-   1 —O—C(O)—O—C₆H₅-   2 —O—C(O)—O—C₆H₄OCH₃-   3 —SH-   4 ═S-   5 —O—CHR²⁴OC(O)—OR²⁵-   6 —O—CHR²⁴—C(O)—R²⁵-   7 —O—CHR²⁴—C(O)—N(R²⁵)₂-   8 —O—CHR²⁴—C(O)—NHR²⁵-   9 —O—CHR²⁴—C(O)—NH₂-   10 —O—CHR²⁴—C(O)—OC₆H₅

Groups 25-1 through 25-10-6. These groups comprise each compound namedin compound groups 1 through 10-6 where R³ substituents 1-10 listed inTable A are replaced with the following groups:

-   1 —O—C(O)—O—C₆H₅-   2 —O—C(O)—O—C₆H₄OCH₃-   3 —SH-   4 ═S-   5 —O—CHR²⁴—C(O)—OR²⁵-   6 —O—CHR²⁴—C(O)—R²⁵-   7 —O—CHR²⁴—C(O)—N(R²⁵⁾ ₂-   8 —O—CHR²⁴—C(O)—NHR²⁵-   9 —O—CHR²⁴—C(O)—NH₂-   10 —O—CHR²⁴—C(O)—OC₆H₅.

Groups 26-1 through 26-25-10-6. These groups comprise each compoundnamed in compound groups 1 through 25-10-6 wherein R⁷ in formula B is—O—, instead of —CH₂—. Thus the 26-1 and 26-2 compounds named 1.2.5.9have the structures

The compound group 26-8-1 and compound group 26-8-2 compounds named1.2.5.9 have the structures

The group 26-11-1 and 26-11-2 compounds named 1.2.5.9 have thestructures

Groups 27-1 through 27-25-10-6. These groups comprise each compoundnamed in compound groups 1 through 25-10-6 wherein R⁸ in formula B is—O—, instead of —CH₂—. Thus the 27-1 and 27-2 compounds named 1.2.5.9have the structures

The group 27-8-1 and group 27-8-2 compounds named 1.2.5.9 have thestructures

The group 27-11-1 and 27-11-2 compounds named 1.2.5.9 have thestructures

Groups 28-1 through 28-25-10-6. These groups comprise each compoundnamed in compound groups 1 through 25-10-6 wherein R⁹ in formula B is—O—, instead of —CH₂— and no double bond is present at the 1-2 position.Thus, there is, e.g., no group 28-3, 28-4, 28-6, 28-8-3, 28-8-4 or28-8-6, since a 1-2 double bond is present in these compounds and a ringoxygen at the 2 position would be charged. The 28-1, 28-2 and 28-5compounds named 1.2.5.9 have the structures

The group 28-8-1 and group 28-8-2 compounds named 1.2.5.9 have thestructures

The group 28-11-1 and 28-11-2 compounds named 1.2.5.9 have thestructures

Groups 29-1 through 29-25-10-6. These groups comprise each compoundnamed in compound groups 1 through 25-10-6 wherein R⁷ is —NH—, insteadof —CH₂—. The compounds are named as described for compound groups 26-1through 26-25-10-6.

Groups 30-1 through 30-25-10-6. These groups comprise each compoundnamed in compound groups 1 through 25-10-6 wherein R⁸ is —NH—, insteadof —CH₂—. The compounds are named as described for compound groups 26-1through 26-25-10-6.

Groups 31-1 through 31-25-10-6. These groups comprise each compoundnamed in compound groups 1 through 25-10-6 wherein R⁹ is —NH—, insteadof —CH₂—. and no double bond is present at the 1-2 position. Thus, thereis e.g., no group 31-3, 31-4, 31-6, 31-8-3, 31-8-4 or 31-8-6. Thecompounds are named as described for compound groups 26-1 through26-25-10-6.

Groups 32-1 through 32-25-10-6. These groups comprise each compoundnamed in compound groups 1 through 25-10-6 wherein two of R⁷, R⁸ and R⁹independently are —NH—, —O— or —S— instead of —CH₂—. The compounds arenamed as described for compound groups 26-1 through 26-25-10-6.

Groups 33-1 through 33-25-10-6. These groups comprise each compoundnamed in compound groups 1 through 25-10-6 wherein each of R⁷, R⁸ and R⁹independently are —NH—, —O— or —S— instead of —CH₂—. The compounds arenamed as described for compound groups 26-1 through 26-25-10-6.

Groups 34-1 through 34-25-10-6. These groups comprise each compoundnamed in compound groups 1 through 25-10-6 wherein R⁷ is —S—, instead of—CH₂—. The compounds are named as described for compound groups 26-1through 26-25-10-6.

Groups 35-1 through 35-25-10-6. These groups comprise each compoundnamed in compound groups 1 through 25-10-6 wherein R⁸ is —S—, instead of—CH₂—. The compounds are named as described for compound groups 26-1through 26-25-10-6.

Groups 36-1 through 36-25-10-6. These groups comprise each compoundnamed in compound groups 1 through 25-10-6 wherein R⁹ is —S—, instead of—CH₂— and no double bond is present at the 1-2 position. There is, e.g.,no group 36-3, 36-4, 36-6, 36-8-3, 36-8-4 or 36-8-6. The compounds arenamed as described for compound groups 26-1 through 26-25-10-6.

Groups 37-1 through 37-25-10-6. These groups comprise each compoundnamed in all of the compound groups 1 through 36-25-10-6 described abovewherein R¹ is not divalent, e.g., is not ═O, and it is in theα-configuration, instead of the β-configuration as shown in formula B.

Groups 38-1 through 38-25-10-6. These groups comprise each compoundnamed in all of the compound groups 1 through 36-25-10-6 described abovewherein R² is not divalent, e.g., is not ═O, and it is in theβ-configuration, instead of the β-configuration as shown in formula B.

Groups 39-1 through 39-25-10-6. These groups comprise each compoundnamed in all of the compound groups 1 through 36-25-10-6 described abovewherein R³ is not divalent, e.g., is not ═O, and it is in theβ-configuration, instead of the α-configuration as shown in formula B.

Groups 40-1 through 40-25-10-6. These groups comprise each compoundnamed in all of the compound groups 1 through 36-25-10-6 described abovewherein R⁴ is not divalent, e.g., is not ═O, and it is in theα-configuration, instead of the β-configuration as shown in formula B.

Groups 41-1 through 41-25-10-6. These groups comprise each compoundnamed in all of the compound groups 1 through 36-25-10-6 described abovewherein R² and R⁴ are not divalent, e.g., they are not ═O, and they areboth in the α-configuration, instead of the β-configuration as shown informula B.

Groups 42-1 through 42-25-10-6. These groups comprise each compoundnamed in all of the compound groups 1 through 36-25-10-6 described abovewherein, when hydrogen is present at the 5-position, it is in theβ-configuration, instead of the α-configuration as shown in formula B.

Any of the compounds or general of compounds that are named in compoundgroups 1 through 42-25-10-6 are suitable for use in the methodsdescribed herein.

In some embodiments, one, or more of R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸independently have the structure(s) and/or independently comprise thenamed compounds, —H, —OH, ═O, —SH, ═S, —NH₂, —CN, —N₃, halogen, ═CH₂,═NOH, ═NOC(O)CH₃, —C(O)—CH₃, —C(O)—(CH₂)₁₋₄—CH₃, —CCH, —CCCH₃, —CH═CH₂,—CH═CH₂CH₃, —O—C(O)—(CH₂)_(m)—(CF₂)_(n)—CH₃,—O—C(O)—(CH₂)_(m)—(CF₂)_(n)—CF₃, —O—C(O)—(CH₂)_(m)—(CF₂)_(n)—CH₂F,—O—C(O)—O—(CH₂)_(m)—(CF₂)_(n)—CH₃, —O—C(O)—O—(CH₂)_(m)—(CF₂)_(n)—CF₃,—O—C(O)—NH—(CH₂)_(m)—(CF₂)_(n)—CH₂F, —O—C(O)—NH—(CH₂)_(m)—(CF₂)_(n)—CH₃,—O—C(O)—NH—(CH₂)_(m)—(CF₂)_(n)—CH₂F₃,—O—C(O)—NH—(CH₂)_(m)—(CF₂)_(n)—CH₂F (where m is 1, 2, 3, 4, 5, 6, 7, 8,9 or 10, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, usually n is 0),—CH(CH₃)—(CH₂)₂—C(O)NH—CH₂COOH, —CH(CH₃)—(CH₂)₂—C(O)NH—CH₂SO₃H,—OSi(CH₃)₂C(CH₃)₃, —C(OH)═CHCH₃, ═CH(CH₂)₀₋₁₅CH₃, —(CH₂)₀₋₁₄CH₂F,—(CH₂)₀₋₁₄CH₂Cl, —(CH₂)₀₋₁₄CH₂Br, —(CH₂)₀₋₁₄CH₂I,—(CH₂)₂₋₁₀—O—(CH₂)₀₋₄CH₃, —(CH₂)₂₋₁₀—S—(CH₂)₀₋₄CH₃,—(CH₂)₂₋₁₀—NH—(CH₂)₀₋₄CH₃, —O—(CH₂)₀₋₁₄CH₂F, —O—(CH₂)₀₋₁₄CH₂Cl,—O—(CH₂)₀₋₁₄CH₂Br, —O—(CH₂)₀₋₁₄CH₂I, —O—(CH₂)₂ ₂₋₁₀—O—(CH₂)₀₋₄CH₃,—O—(CH₂)₂₋₁₀—S—(CH₂)₀₋₄CH₃, —O—(CH₂)₂₋₁₀—NH—(CH₂)₀₋₄CH₃,—O—C(O)—(CH₂)₀₋₁₄CH₂F, —O—C(O)—(CH₂)₀₋₁₄CH₂Cl, —O—C(O)—(CH₂)₀₋₁₄CH₂Br,—O—C(O)—(CH₂)₀₋₁₄CH₂I, —O—C(O)—(CH₂)₂₋₁₀CH₃—O—(CH₂)₀₋₄CH₃,—O—C(O)—(CH₂)₂₋₁₀—S—(CH₂)₀₋₄CH₃, —O—C(O)—(CH₂)₂₋₁₀—NH—(CH₂)₀₋₄CH₃,—O—C(S)—(CH₂)₀₋₁₄CH₂F, —O—C(S)—(CH₂)₀₋₁₄CH₂Cl, —O—C(S)—(CH₂)₀₋₁₄CH₂Br,—O—C(S)—(CH₂)₋₁₄CH₂I, —O—C(S)—(CH₂)₂₋₁₀—O—(CH₂)₀₋₄CH₃,—O—C(S)—(CH₂)₂₋₁₀—S—(CH₂)₀₋₄CH₃, —O—C(S)—(CH₂)₂₋₁₀—NH—(CH₂)₀₋₄CH₃,—(CH₂)₀₋₁₆NH₂, —(CH₂)₀₋₁₅CH₃, —(CH₂)₀₋₁₅CH═CH₂, —(CH₂)₀₋₁₅NHCH(O),—(CH₂)₀₋₁₆NH—(CH₂)₀₋₁₅CH₃, —(CH₂)₀₋₁₅CCH, —(CH₂)₀₋₁₅OC(O)CH₃,—(CH₂)₀₋₁₅OCH(OH)CH₃, —(CH₂)₀₋₁₅C(O)OCH₃, —(CH₂)₀₋₁₅C(O)OCH₂CH₃,—(CH₂)₀₋₁₅C(O)(CH₂)₀₋₁₅CH₃, —(CH₂)₀₋₁₅C(O)(CH₂)₀₋₁₅CH₂OH,—O(CH₂)₁₋₁₆NH₂, —O(CH₂)₁₋₁₅CH₃, —O(CH₂)₁₋₁₅CN, —O(CH₂)₁₋₁₅CH═CH₂,—O(CH₂)₁₋₁₅NHCH(O), —O(CH₂)₁₋₁₆NH—(CH₂)₁₋₁₅CH₃, —O(CH₂)₁₋₁₅CCH,—O(CH₂)₁₋₁₅OC(O)CH₃, —O(CH₂)₁₋₁₅OCH(OH)CH₃, —O(CH₂)₁₋₁₅C(O)OCH₃,—O(CH₂)₁₋₁₅C(O)OCH₂CH₃, —O(CH₂)₁₋₁₅C(O)(CH₂)₀₋₁₅CH₃,—O(CH₂)₁₋₁₅C(O)(CH₂)₀₋₁₅CH₂OH, —OC(O)(CH₂)₁₋₁₆NH₂, —OC(O)(CH₂)₁₋₁₅CH₃,—C(O)O(CH₂)₁₋₁₅CN, —C(O)O(CH₂)₁₋₁₅CH═CH₂, —OC(O)(CH₂)₁₋₁₅NHCH(O),—OC(O)(CH₂)₁₋₁₈NH—(CH₂)₁₋₁₅CH₃, —OC(O)(CH₂)₁₋₁₅CCH,—OC(O)(CH₂)₁₋₁₅OC(O)CH₃, —OC(O)(CH₂)₁₋₁₅OCH(OH)CH₃,—OC(O)(CH₂)₁₋₁₅C(O)OCH₃, —OC(O)(CH₂)₁₋₁₅C(O)OCH₂CH₃,—OC(O)(CH₂)₁₋₁₅C(O)(CH₂)₀₋₁₅CH₃, —OC(O)(CH₂)₁₋₁₅C(O)(CH₂)₀₋₁₅CH₂OH,phosphoenolpyruvate, D-glucosamine, glucholic acid, glucuronic acid,pantothenic acid, pyruvic acid, glucose, fructose, mannose, sucrose,lactose, fucose, rhamnose, galactose, ribose, 2′-deoxyribose,3′-deoxyribose, glycerol, 3-phosphoglycerate, a PEG (PEG 20, PEG 100,PEG 200, PEG 10000), a polyoxyalkylene polymer, glycine, alanine,phenylalanine, threonine, proline, 4-hydroxyproline or anoligonucleotide or analog that comprises about 4 to about 21 monomers.

When a substituent is an oligonucleotide or a polymer usually only a oneof these is bonded to the formula 1 compound. Typically, when R¹-R² andR⁴-R⁶ comprise one or more of these substituents (or others describedherein), the substituent is present in the β-configuration, while R³typically comprises a substituent in the β-configuration. In someembodiments, R² is in the α-configuration.

In some embodiments, one or more of R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸independently comprise a nucleoside, a nucleotide, an oligonucleotide oran analog of any of these moieties. Typically such moieties are linkedto the steroid nucleus through a terminal hydroxyl, thiol, acyl moietyor amine at the 5′, 3′ or 2′ positions, when a hydroxyl, thiol, acylmoiety or amine is present at that position. For oligonucleotides andoligonucleotide analogs, the linkage to the steroid occasionally isthrough a sugar hydroxyl at an internal 2′ position.

Analogs of phosphodiester linkages include phosphorothioate linkages andothers as described in the cited references. Oligonucleotide couplinggroups means any moiety suitable for generating a phosphodiester linkageor phosphodiester analog linkage between adjacent nucleotides or theiranalogs. Suitable oligonucleotide coupling groups include —OH,H-phosphonate, alkylphosphonamidites or phosphoramidites such asβ-cyanoethyl-phosphoramidite,N,N-diisopropylamino-β-cyanoethoxyphosphine and others as described inthe cited references. Suitable purine and pyrimidine bases includeadenine, guanine, cytosine, thymine, uracil and others as described inthe cited references. Suitable nucleosides, nucleotides,oligonucleotides and their analogs have been described, see e.g., U.S.Pat. Nos. 4,725,677, 4,973,679, 4,997,927, 4,415,732, 4,458,066,5,047,524, 4,959,463, 5,212,295, 5,386,023, 5,489,677, 5,594,121,5,614,622, 5,624,621; and PCT publication Nos. WO 92/07864, WO 96/29337,WO 97/14706, WO 97/14709, WO 97/31009, WO 98/04585 and WO 98/04575 allof which are incorporated herein by reference. The formula 1 compounds,e.g., those named in any of the compound groups 1 through 42-25-10-6,are suitable for linkage to oligonucleotides modulate the lipophilicityof oligonucleotides or the transport or permeation of an oligonucleotideinto cells. Such linkages may be biologically labile to facilitaterelease of the steroid from the oligonucleotide once the conjugate hasentered the cell.

Table 2 shows these and other exemplary moieties that one or more ofR¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸ independently can comprise. Pr means aprotecting group. These moieties are often bonded to one or more of theR¹, R² and R⁴ positions, usually to one or two of those positions. Forstructures with more than one of a given variable, e.g., X in structureA3 or A5, each is independently selected.

TABLE 2

G ═CH₂

Typical containers for storage of the invention compositions andformulations will limit the amount of water that reaches the materialscontained therein. Typically, formulations are packaged in hermeticallyor induction sealed containers. The containers are usually inductionsealed. Water permeation characteristics of containers have beendescribed, e.g., Containers—Permeation, chapter, USP 23 <671>, UnitedStates Pharmacopeial Convention, Inc., 12601 Twinbrook Parkway,Rockville, Md. 20852, pp.: 1787 et seq. (1995).

The use of formula A compounds for treatment of certain diseases, e.g.,infections such as malaria, HCV or Cryptosporidium, has been described.Formula A compounds have the structure

where Q₁ is —C(R₁)₂— or —C(O)—; Q₂ is —C(R₁)₂—, —C(R₁)(Y)—, —C(Y)— or—CH₂—CH₂—; Q₃ is —H or —C(R₁)₃—; Q₄ is —C(R₁)₂—, —C(O)—,hydroxyvinylidine (—CH(CH═CHOH)—) or methyl methylene (—CH(CH)₃—); Q₅ is—C(R₁)₂— or —C(O)—; X and Y independently are —OH, —H, lower alkyl(e.g., C₁₋₆ alkyl), —O—C(O)—R₅, —C(O)—OR₅, halogen (i.e., —F, —Cl, —Bror —I) or ═O; each R₁ independently is —H, —F, —Cl, —Br, —I, —OH, C₁₋₆alkoxy, or C₁₋₆ alkyl; R₂ is —H, —OH, —F, —Cl, —Br, —I, C₁₋₆ alkyl, C₁₋₆alkoxy, —OR₃, an ester (e.g., —O—C(O)—R₄ or —C(O)—O—R₄), a thioester(e.g., —O—C(S)—R₄ or —C(S)—O—R₄), a thioacetal (e.g., —S—C(O)—R₄, or—C(O)—S—R₄), a sulfate ester (e.g., —O—S(O)(O)—O—R₄), a sulfonate ester(e.g., —O—S(O)—O—R₄) or a carbamate (e.g., —O—C(O)—NH—R₄ or—NH—C(O)—O—R₄) or R₂, together with the R₁ that is bonded to the samecarbon atom is ═O; R₃ is —S(O)(O)—OM,—S(O)(O)—O—CH₂—CH(O—C(O)—R₆)—CH₂—O—C(O)—R₆,—P(O)(O)—O—CH₂—CH(O—C(O)—R₇)—CH₂—O—C(O)—R₇, a glucuronide group ofstructure (B)

or R₃ is C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, a C₁₋₁₈ ester or aC₁₋₁₈ thioester, where any of the foregoing C₁₋₁₈ or C₂₋₁₈ moieties areoptionally substituted at one or more hydrogen atoms with one or moreindependently selected —OR^(PR), (including —OH), —NHR^(PR), (including—NH₂) or —SR^(PR), (including —SH) groups, or R₃ is a C₁₋₁₈ fatty acid,C₂₋₁₀ alkynyl, (J)_(n)-phenyl-C₁₋₅-alkyl, (J)_(n)-phenyl-C₂₋₅-alkenyl;R₄ is —H, a protecting group, optionally substituted C₁₋₁₈ alkyl,optionally substituted C₁₋₁₈ alkenyl, optionally substituted C₁₋₁₈alkynyl, optionally substituted aryl, optionally substituted aryl-C₁₋₆alkyl, optionally substituted aryl-C₂₋₆ alkenyl, optionally substitutedaryl-C₂₋₆ alkynyl, optionally substituted heterocycle-C₁₋₆ alkyl,optionally substituted C₂₋₆ alkenyl-heterocycle, optionally substitutedC₂₋₆ alkynyl-heterocycle or an optionally substituted heterocycle, whereany of the foregoing moieties are optionally substituted at one, two,three, four, five or more carbon or hydrogen atoms with one or moreindependently selected —O—, —S—, —NR^(PR)— (including —NH—), —NH—C(O)—,—OR^(PR) (including —OH), —NHR^(PR) (including —NH₂), —SR^(PR)(including —SH), ═O, ═S, ═N—OH, —CN, —NO₂, —F, —Cl, —Br or —I groups oratoms; each R₅ independently is straight or branched C₁₋₁₄ alkyl; eachR₆ independently is straight or branched C₁₋₁₄ alkyl; each R₇independently is straight or branched C₁₋₁₄ alkyl or a glucuronide groupof structure (B); each R^(PR) independently is —H or an independentlyselected protecting group; n is 0, 1, 2 or 3; each J independently is—F, —Cl, —Br, —I, C₁₋₄ alkyl, C₁₋₄ alkenyl, C₁₋₄ alkoxy, carboxy, nitro,sulfate, sulfonyl, a C₁₋₆ carboxyl ester or a C₁₋₆ sulfate ester; M ishydrogen, sodium, —S(O)(O)—O—CH₂——CH(O—C(O)—R₆)—CH₂—O—C(O)—R₆,—P(O)(O)—O—CH₂—CH(O—C(O)—R₇)—CH₂—O—C(O)—R₇ or a glucuronide group ofstructure (A); the dotted lines in formula 1 represent an optionaldouble bond, provided that there are not double bonds at both the 4-5and 5-6 positions and provided that when a double bond is present, zeroor 1 R₁ group is bonded to carbon atoms at the 1-, 2-, 4-, 5-, 6- or 17positions so that these carbon atoms are tetravalent; and the salts,stereoisomers, positional isomers, metabolites, analogs or precursors.

The formula A compounds, particularly where both R₁ at the 11-positionare not hydroxyl, alkoxy or a moiety that can hydrolyze to a hydroxyl,are generally suitable for use in the methods and compositions that aredisclosed herein, e.g., their use to enhance a subject's Th1 immuneresponses. Methods of administration and dosages are essentially asdescribed herein.

Intermittent dosing methods. One can intermittently administer theformula 1 compound(s), e.g., BrEA or a BrEA ester, to a subject withoutsome of the undesired aspects normally associated with discontinuousdosing. Such undesired aspects include development of resistance of apathogen (virus such as HIV or a parasite such as a Plasmodium parasite)to the therapeutic agent or failure of the patient or subject to adhereto a dosing regimen. Intermittent dosing protocols includeadministration of a formula 1 compound, e.g., orally, topically orparenterally as follows: (1) dosing for about 3 to about 20 days, (2) nodosing of the formula 1 compound for about 4 to about 20 days, (3)dosing for about 4 to about 20 days and (4) optionally repeating thedosing protocol 1, 2, 3, 4, 5, 6, 10, 15, 20, 30 or more times. Often,the dosing of steps (1) and (3) will be maintained for about 3-15 days,usually about 3-5 days. In general, steps (1)-(3) of the dosing protocolrecited above, will be repeated at least one time, typically at least 2,3, 4, 5 or 6 times. For infections that tend to remain chronic, e.g.,HIV, HCV or other chronic virus or parasite infection, the intermittentdosing protocol is typically maintained over a relatively long timeperiod, e.g., for at least about 6 months to about 5 years.

In these intermittent dosing protocols, the formula 1 compound(s) can beadministered by any suitable route, e.g., intramuscular (i.m. or I.M.),subcutaneous (s.c. or S.C.), intravenous (i.v. or I.V.), intradermal,other parenteral route, aerosol using about 0.1 to about 10 mg/kg/day,usually about 0.2-4 mg/kg/day. Alternatively, one can administer theformula 1 compound(s) orally using about 4 to about 40 mg/kg/day,usually about 6-20 mg/kg/day. In some embodiments, the intermittentdosing methods exclude dosing protocols that are commonly used todeliver contraceptive steroids to, e.g., human females, such as dailydosing for 21 days, followed by no dosing for 7 days. In general, thenon-aqueous formulations described herein that contain formula 1compound(s) are administered i.m. or s.c., while aqueous formulationsthat contain formula 1 compound(s) is administered by i.v., i.m., s.c.or other parenteral routes. The daily doses can be administered as asingle dose, especially for doses given parenterally, or the dose can besubdivided into two, three or four subdoses, usually two, especially fordoses given orally.

Exemplary embodiments are (a) administering a formula 1 compound(s),e.g., BrEA or an ester or carbonate of BrEA, once every other day for 20days, followed by (b) no dosing for 1, 2, 3, 4, 5, 6, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60 or more days and then (c) administering theformula 1 compound(s) at least once more on one day, e.g., administeringthe formula 1 compound(s) once every other day for 20 days and (d)optionally repeating (a), (b) and (c) 1, 2, 3, 4, 5 or 6 times or more.A subset of these embodiments are (a) administering a formula 1compound(s), e.g., BrEA or an ester or carbonate of BrEA, once everyother day for 20 days, followed by (b) no dosing for about 10-40 daysand then (c) administering the formula 1 compound(s) at least once moreon one day, e.g., administering the formula 1 compound(s) once everyother day for 20 days and (d) optionally repeating (a), (b) and (c) 1,2, 3, 4, 5 or 6 times or more. In any of these embodiments, one canadminister the formula 1 compound(s) in 2 or 3 subdoses per day.

Other embodiments are (a) administering a formula 1 compound(s), e.g.,BrEA or an ester or carbonate of BrEA, once every day for about 8-12days, followed by (b) no dosing for 1, 2, 3, 4, 5, 6, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60 or more days and then (c) administering theformula 1 compound(s) at least once more on one day, e.g., administeringthe formula 1 compound(s) once per day for about 8-12 days and (d)optionally repeating (a), (b) and (c) 1, 2, 3, 4, 5 or 6 times or more.A subset of these embodiments are (a) administering a formula 1compound(s), e.g., BrEA or an ester or carbonate of BrEA, once every dayfor about 10 days, followed by (b) no dosing for about 10-40 days andthen (c) administering the formula 1 compound(s) at least once more onone day, e.g., administering the formula 1 compound(s) once per day forabout 10 days and (d) optionally repeating (a), (b) and (c) 1, 2, 3, 4,5 or 6 times or more. In any of these embodiments, one can administerthe formula 1 compound(s) in 2 or 3 subdoses per day.

One aspect of invention intermittent dosing is monitoring the subject'sresponse to dosing. For example, while dosing a subject who has a viralinfection (e.g., HCV, HIV, SIV, SHIV), one can measure the subject's orpathogen's response, e.g., amelioration of one or more symptoms or achange in infectious particles or viral RNA in the serum. Once aresponse is observed dosing can be continued for one, two or threeadditional days, followed by discontinuing the dosing for at least oneday (at least 24 hours), usually for at least 2 or 3 days. Once thesubject's response shows signs of remission (e.g., viral serum RNAbegins to increase), dosing can be resumed for another course. An aspectof the subject's response to formula 1 compound(s) is that the subjectmay show a measurable response within a short time, usually about 5-10days, which allows straightforward tracking of the subject's response,e.g., by monitoring viral titer in peripheral white blood cells (“PBMC”)or by measuring viral nucleic acid levels in the blood. One may monitorone or more immune cell subsets, e.g., NK, LAK, dendritic cells or cellsthat mediate ADCC immune responses, during and after intermittent dosingto monitor the subject's response and to determine when furtheradministration of the formula 1 compound is indicated. These cellsubsets are monitored as described herein, e.g., by flow cytometry.

For any of the treatments or methods described herein, prolongedbeneficial effects or a sustained immune response by a subject mayresult from a single administration or a few daily administrations ofthe formula 1 compound for from intermittent treatment with the formula1 compound. A single administration means that a formula 1 compound isadministered to the subject in one, two, three or more doses within a 24hour period and no further administration of any formula 1 compound tothe subject occurs for at least about 45 days to about 2 months, e.g.,for 3, 4, 5, 6 or more months. Prolonged beneficial effects or immuneresponses may also persist after a short course of treatment has beencompleted (e.g., daily dosing for 2, 3, 4, 5 or 6 days) and the subjectis no longer receiving any formula 1 compound, or, in some cases, anyother therapeutic treatment to treat the primary cause of the subject'spathological condition. Such beneficial effects can persist for morethan about 5-30 days.

In some cases, beneficial effects from treatment have been observed formore than 3 months (4 or 5 or more months) after a short course oftreatment of a subject with a formula 1 compound. Thus, administrationof a formula 1 compound provides a method to effectively protect asubject against progression of an infection or against adverseconsequences of unwanted immune reactions (e.g., inflammation) oragainst immunosuppression (from infection, chemotherapy, etc), withoutany dosing of the compound for at least 3 months after an initial dosingprotocol, which could be an intermittent or a continuous dosing protocolover, e.g., 1 day to about 4 months (1-15 days, about 1 month, about 2months, etc).

Synthesis methods. Reagents and reaction conditions that one can use tomake the formula 1 compounds have been described, see e.g., thecitations above, U.S. Pat. Nos. 5,874,598, 5,874,597, 5,874,594,5,840,900; PCT publication number WO 9901579. General chemical syntheticmethods to link a variety of organic moieties to various reactive groupshave been described. For example, in G. T. Hermanson, BioconjugateTechniques, Academic Press, 1996, functional targets such as aminoacids, peptides and carbohydrates are described at pages 3-136, whilethe chemistries of reactive groups in the functional targets, e.g.,amine, thiol, carboxyl, hydroxyl, aldehyde, ketone and reactive hydrogenatoms (e.g., —H linked to an electron-donating moiety such as aheteroaryl moiety) are described at pages 137-166. This reference alsodescribes reagents useful to make the derivatives, e.g., zero-lengthcross-linkers, heterobifunctional cross-linkers, homobifunctionalcross-linkers, tags, probes and polymers are described at pages 169-416and 605-638. This reference also describes synthetic methods to modifyoligonucleotides at pages 639-671.

In one aspect, amino acids or peptides are linked to the steroid throughthe amine group using a coupling reagent such as phosgene (Cl—CO—Cl) orCl—CS—Cl and suitably protected amino acids or peptides and steroids,which are protected as needed. Such linkage generates an intervening—CO—O— or a —CS—O— moiety between the amino acid or peptide and thesteroid nucleus.

Exemplary synthesis schemes. By way of exemplification and notlimitation, the following methods are used to prepare the one or more ofthe compounds disclosed herein. Starting materials and straightforwardvariations of the schemes are found, e.g., in the following references,which are incorporated herein by reference: A. P. Davis, et al.,Tetrahedron Lett., 33: 5111-5112, 1992; I. Takashi, et al., Chem. Pharm.Bull., 34: 1929-1933, 1986; I. Weisz, et al., Arch. Pharm., 319:952-953, 1986; T. Watabe, et al., J. Med. Chem., 13: 311-312, 1970; M.Davis, et al., J. Chem. Soc. C., (11): 1045-1052, 1967; R. C. Cambie, etal., J. Chem. Soc., Perkin Trans. 1, (20): 2250-2257, 1977; L. Minale,et al., J. Chem Soc., Perkin Trans. 1, (20): 2380-2384, 1974; C. K. Lai,et al., Steroids, 42: 707-711, 1983; S. Irie, et al., Synthesis, (9):1135-1138, 1996; E. J. Corey, J. Am. Chem. Soc., 118: 8765-8766, 1996;M. E. Annunziato, et al., Bioconjugate Chem., 4: 212-218, 1993; N. J.Cussans, et al., J. Chem. Soc., Perkin Trans. 1, (8): 1650-1653, 1980;D. H. R. Barton, et al., J. Chem. Soc., Chem. Commun., (9): 393-394,1978; H. Loibner, et al., Helv. Chim. Acta, 59: 2100-2113, 1976; T. R.Kasturi, et al., Proc. Indian Acad. Sci., [Ser.]: Chem. Sci., 90:281-290, 1981; T. Back, J. Org. Chem., 46: 1442-1446, 1981; A. Canovas,et al., Helv. Chim. Acta, 63: 486-487, 1980; R. J. Chorvat, et al., J.Org. Chem., 43: 966-972, 1978; M. Gumulka, et al., Can. J. Chem., 63:766-772, 1985; H. Suginome, et al., J. Org. Chem., 55: 2170-2176, 1990;C. R. Engel, et al., Can. Heterocycles, 28: 905-922, 1989; H. Sugimone,et al., Bull, Chem. Soc. Jpn., 62: 193-197, 1989; V. S. Salvi, et al.,Can. Steroids, 48: 47-53, 1986; C. R. Engel, et al., Can. Steroids, 47:381-399, 1986; H. Suginome, et al., Chem. Lett., (5): 783-786, 1987; T.Iwadare, et al., J. Chem. Soc., Chem. Commun., (11): 705-706, 1985; H.Nagano, et al., J. Chem. Soc., Chem. Commun., (10):; 656-657, 1985; V.S. Salvi, et al., Steroids, 27: 717-725, 1976; C. H. Engel, et al,Steroids, 25: 781-790, 1975; M. Gobbini, et al., Steroids, 61: 572-582,1996; A. G. Gonzalez, et al., Tetrahedron, 46: 1923-1930, 1990; S. C.Bobzin, et al., J. Org. Chem., 54: 3902-3907, 1989; B. Solaja, et al.,Croat. Chem. Acta, 59: 1-17, 1986; Y. Kashman, et al., Tetrahedron, 27:3437-3445, 1971; K. Yoshida, et al., Chem. Pharm. Bull. (Tokyo), 15:1966-1978, 1967; P. B. Sollman, et al., Chem. Commun. (11): 552-554,1967; H. Suginome, et al., J. Org. Chem., 55: 2170-2176, 1990; H.Suginome, et al., Journal Chem. Lett., (5): 783-786, 1987; G. A.Tolstikov, et al., Zh. Org. Khim., 22: 121-132, 1986; T. Terasawa, etal., J. Chem. Soc., Perkin Trans. 1, (4): 990-1003, 1979; Z. Zhuang, etal., Yougi Huaxue, (4): 281-285, 1986; W. T. Smith, et al., Trans. Ky.Acad. Sci., 45: 76-77, 1984; A. K. Batta, et al., Steroids, 64: 780-784,1999; B. Ruan, et al., Steroids, 65: 29-39, 2000; L. Garrido, et al.,Steroids, 65: 85-88, 2000; P. Ramesh, et al., Steroids, 64: 785-789,1999; M. Numazawa, et al., Steroids, 64: 187-196, 1999; P. N. Rao, etal., Steroids, 64: 205-212, 1999; M. Numazawa, et al., Steroids, 64:320-327, 1999; U.S. Pat. Nos. 3,281,431, 3,301,872, 3,325,535,3,325,536, 3,952,018, 4,602,008, 5,571,795, 5,627,270, 5,681,964,5,744,453; international publication numbers WO 9408588, WO 9508558, WO9508559, WO 9638466, WO 9809450; United Kingdom patent numbers GB1168227, GB 813529, GB 802618; French patent number 824529; Japan patentnumber JP 45010134; European patent applications EP 232788, EP 430078;and German patent number DE 19631189.

Scheme 1. For the structures shown in scheme 1, R⁵-R⁹ are as defined forformula 1 compounds. Thus, when R⁵ and R⁶ are both —CH₃ in theβ-configuration, R⁷, R⁸ and R⁹ are all —CH₂—, H at the 9 and 14positions are in the α-configuration, acetate at the 3-position is inthe β-configuration, and H at the 8 position is in the β-configuration,the first compound in scheme 1 is DHEA acetate. The acetate groups atthe 3, 7, 16, 17 or other positions in this scheme and in other schemesdisclosed herein may independently be other ester moieties as describedherein, e.g., C₂₋₅₀ esters including —C(O)—(CH₂)₀₋₄—(CF₂)₀₋₄—CF₃,including —C(O)—CF₃, —C(O)—C₂₋₂₉ optionally substituted alkyl,—C(O)—CH₂—C₂₋₂₈ optionally substituted alkenyl, —C(O)—CH₂—C₂₋₂₈optionally substituted alkynyl, —C(O)—(CH₂)₀₋₆-optionally substitutedphenyl, or —C(O)—(CH₂)₀₋₆-optionally substituted heterocycle or otherorganic moieties as disclosed herein or in the cited references.

Typical substituents for these organic moieties are as described herein,including one, two, three or more independently selected —O—, ═O,optionally protected hydroxyl, —S—, optionally protected thiol, —NH—,optionally protected —NH₂, optionally protected —C(O)OH, —C(O)—NH—,—C(O)—NH₂, —NH₂—C(O)—H, —NH₂—C(O)—C₀₋₄H₁₋₉, —NH₂——C(O)—O—C₀₋₄H₁₋₉, —CN,—NO₂, —N₃ or halogen. Reactive groups are protected as needed, e.g., ═Owould usually be protected in the LiCR reaction that is used to generatecompound 1 in scheme 1 below.

Abbreviations:

-   LDA=lithium diisopropyl amide; MCPBA=m-chloroperbenzoic acid;    TMSCI=trimethychlorosilane; DMAP=4-dimethylaminopyridine;    Dibromantin=1,3-dibromo-4,4-dimethylhydantoin.-   R═CR^(A); R^(A)═—H or a C1-C50 organic moiety as described herein,    e.g., —H, —C₁₋₂₀ optionally substituted alkyl, —C₁₋₂₀ optionally    substituted alkenyl, —C₁₋₂₀ optionally substituted alkynyl,    —(CH₂)₀₋₆-optionally substituted phenyl or —(CH₂)₀₋₆-optionally    substituted heterocycle.

Scheme 2. Compounds of formula 2 are prepared from structure A compoundsshown in scheme 1 using the last two steps of Scheme 1: (1) dibromantin,(2) LiBr, (3) Li—C—R, where R is CR^(A) and R^(A) is —H or —C₁₋₁₂optionally substituted alkyl. When R⁷, R⁸ and R⁹ are all —CH₂—, H at the9 and 14 positions are in the α-configuration and H at the 8 position isin the β-configuration the first compound in scheme 1 is DHEA acetate.Typical substituents for the R^(A) alkyl moiety includes one, two ormore independently selected —O—, optionally protected ═O, optionallyprotected hydroxyl, —S—, optionally protected thiol, —NH—, optionallyprotected —NH₂, optionally protected —C(O)OH, —C(O)—NH—, —C(O)—NH₂,—NH₂—C(O)—H, —NH₂—C(O)—C₀₋₄H₁₋₉, —NH₂—C(O)—O—C₀₋₄H₁₋₉, —CN, —NO₂, —N₃ orhalogen.

Scheme 3. The allylic bromination at C-7 is done as in Scheme 1. R andR^(A) are as defined in Schemes 1 and 2.

Scheme 4. The addition of lithium reagent (lithium acetylide when R is—CH) to the 17-position >C═O in the presence of the bromide at C-16results in epoxide formation or in a pinacol rearrangement.Alternatively, compounds without of structure 3 can be dehydrated bymild acid catalysis to form compounds of formula 4 by treatment of thealkene with Br₂, H₂O. R and R^(A) are as defined in Schemes 1 and 2.

Scheme 5. Sodium borohydride gives a mixture of epimers at C-7, whichmay be separated by standard methods, e.g., HPLC, TLC or columnchromatography. To obtain the pure 7α-OH compound, allylic brominationfollowed by hydrolysis is accomplished as described in Schemes 1 and 3.

Scheme 6. Formula 6 compounds are prepared by treatment of the acetatewith lithium acetylide as in Schemes 1, 2, 3 or 4. R and R^(A) are asdefined in Schemes 1 and 2.

Scheme 7. Formula 7 compounds are prepared from the 3-acetate withreagents described in Schemes 1 and 4. R and R^(A) are as defined inSchemes 1 and 2.

Scheme 8. Formula 8 compounds are prepared from the formula A compoundsby sodium borohydride reduction at C-17 followed by acetylation.

Scheme 9. The starting material is made using reactions described inSchemes 1 and 3.

Scheme 10. Reduction and acetylation at C-3 and hydrolysis and oxidationat C-17 will allow formula 10a and 10b compounds to undergofunctionalization as shown in Schemes 1-9 at C-3, C-16 and C-17. The7-oxo acetate can be substituted for the formula A compound 3-acetateand functionalization at C-3, C-16 and C-17 is achieved similarly for7-oxo compounds using the reactions shown in schemes 1-9.

Treatment of 10a with LDA, followed by alkylation of the enolate allowsintroduction of side chains such as R¹⁰, which may be, e.g., C1-C20alkyl (methyl, ethyl), C1-C20 alkenyl (CH₂═CH—(CH₂)₀₋₆—), benzyl,—(CH₂)₁₋₄—O—(CH₂)₀₋₄—CH₃.

Schemes 1-9 show the introduction of the hydroxyl function at thepositions shown. Methods to convert hydroxyl to other functional groupsare accomplished essentially as described, e.g., in the references citedherein. For example, esters, of formula 1-10c compounds, such as—O—C(O)—R^(B) where R^(B) is a C₁₋₅₀ organic moiety, are prepared fromthe steroid alcohol by treatment with the appropriate acid anhydride oracid chloride (R^(B)—C(O)—Cl) to form any desired ester. Ethers, such as—O—R^(B), are prepared from alcohols by formation of the alkaline metalalkoxide (Na⁺ or K⁺) followed by treatment with a primary or secondaryiodide (R^(B)—I). Thionoesters, R^(B)—C(S)—O—, are prepared by treatingthe R^(B)—C(O)—O— ester with Lawesson's reagent.

Sulfates, NaO—S(O)(O)—O—, R^(B)—O—S(O)(O)—O—, e.g.,CH₃(CH₂)₀₋₁₈—S(O)(O)—O—, are prepared by treatment of alcohols withchlorosulfonic acid followed by NaOH or alternatively by oxidation ofsulfites using kMnO₄. If the alkyl (e.g., methyl) ester is desiredalkylchloro-sulfonate (methylchloro-sulfonate) can be used. SulfitesHO—S(O)—O— and ammonium salts NH₄ O—S(O)—O, or R^(B) O—S(O)—O— esters(e.g., CH₃ O—S(O)—O—) are prepared by standard methods. The ammoniumsalts are prepared by treatment of alcohols with ammonia and sulfurdioxide. The esters such as alkyl, alkenyl and alkynyl esters (e.g.,methyl ester) are obtained when alcohols are treated withalkylchlorosulfite (e.g., methycholorosulfite), alkenylchlorosulfite oralkynylchlorosulfite in the presence of a suitable base such astriethylamine. Phosphoesters, R^(B)O—P(OR^(PR))(O)—O— are prepared bytreating the alcohol with diethylchlorophosphate in the presence ofNa₂CO₃. Alternatively, if the alcohol is treated with phosphoric aciddiesters in the presence of triphenylphospine (PPh₃) anddiethylazodicaboxylate (DEAD) the corresponding triesters are formedwith inversion (Mitsunobu reaction).

Phosphothioesters, R^(B)—O—P(SR^(PR))(O)—O— are generated by treatmentof alcohols with the monothio analog of diethylchlorophosphate asdescribed for phosphoesters yielding the phosphothioesters. Carbonates,R^(B)—O—C(O)—O— are generated from the corresponding steroid alcoholusing the chloroformate (R^(B)—C(O)—Cl), e.g., C₁₋₂₀ alkyl, alkenyl oralkynyl chioroformates (e.g. CH₃(CH₂)₀₋₅—C(O)Cl). Carbamates,R^(B)—NH—C(O)—O— are made from steroid alcohols by treatment withisocyanates (R^(B)N═C═O) or NaOCN in the presence of trifluroroaceticacid. Amino acid esters, ZNX—CHY—C(O)—O— are generated by coupling thesteroid alcohol with the acid chloride of the N-protected amino acid.

Oxidation of hydroxyl groups that are linked to the steroid nucleus isused to obtain ketones and related functionalities. For example,conversion of alcohols to ketones can be achieved using a variety ofoxidizing agents such as CrO₃ in AcOH, or pyridinium cholorchromate,pyridinium dichromate or oxalyl chloride with triethylamine (Swernoxidation). Thioketones (═S) are prepared by treating ketones withLawesson's reagent(2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide;commercially available from Aldrich). Thioacetals, —C(SR^(B))(SR^(B))—,are prepared from ketones (—C(O)—) by treatment with R^(B)—SH thiolsunder acid catalysis conditions (e.g., HCl). Phosphonoesters,RO—P(OR^(PR))(O)—, are generated by addition of the phosphorus aciddiester to ketones in the presence of KF to yield hydroxyphosphonoesters. One may optionally remove the hydroxy group using adehydration and hydrogenation sequence.

Substitution of hydroxyl groups is used to generate a number offunctionalities. For example, thiols, —SH, are prepared from alcohols byconversion of the alcohol with inversion to the bromide using PBr₃.Treatment of the bromide with thiourea followed by NaOH gives the thiol.Thioethers, R^(B)—S—, are prepared from thiols by treatment with NaOHand the required halide, e.g., alkyl halide. Alternatively, alcoholderivatives like tosylates or mesylates can be displaced by thiolateanions, R^(B)—S⁻, to yield the thioether. Thioesters, R—C(O)—S—, areprepared by treating the tosylate (mesylate) of the alcohol with thesodium salt of the thioacid.

Substitution of hydroxyl groups can be used to generate both esters,R^(B)O—C(O)—, and amides, NHR^(B)—C(O)—, linked to the steroid at carbonatoms. For amides and amines, R^(B) is —H, a protecting group or a C₁₋₅₀organic moiety. These are synthesized from the steroid bromide withinversion by displacement with NaCN. The cyanide group can be hydrolyzedto the amide or the acid. The acid is esterified or treated by standardpeptide coupling reactions with an O-protected amino acid in thepresence of a suitable carboxyl activating agents such asdicyclohexylcarbodiimide (DCC) to form steroid —C(O)—NH—CHY—C(O)—OR,where Y is the side chain of an amino acid or a C1-C10 organic moietyand R is a protecting group (or hydrogen when deprotected).

Amines and derivatives of amines, e.g., R^(B)NH—, R^(B)—C(O)NH—,R^(B)OC(O)—NH— or R^(B)O—C(O)—CHR^(B)—NH— linked to steroid carbonatoms, are typically prepared by standard methods. For example, amines(NH₂-steroid) are generally prepared using the Hoffmann rearrangement(Br2, NaOH) from the amide (NH₂—C(O)-steroid) or the Curtiusrearrangement (NaN₃) from the acid chloride of the steroid. The R^(B)substituent can subsequently be introduced by alkylation. Steroidalcohols can be used as starting materials under standard Mitsunobuconditions (PPh₃, DEAD) to yield N-Boc sulfonamides usingN-(t-butoxycarbonyl)-p-toluenesulfonamide. One can selectively removeeither protecting group. Treatment with trifluoroacetic acid affords thesulfonamide (R₆—S(O)(O)—NH-steroid). Alternatively, sodium napthalenidedeprotects to give the N-Boc compound. Amines (NH₂-steroid) can beconverted to amides (R^(B)NH—C(O)-steroid) using acyl chlorides(R^(B)—C(O)—Cl). Treatment with ethyl chloroformate gives theN-carbamate (R^(B)—OC(O)—NH-steroid). The amine (NH₂-steroid) can bealkylated with an α-bromoester (R^(B)—C(O)—CHY—NH₂) to yield the aminoacid substituted steroid (R^(B)—O—C(O)—CHY—NH-steroid).

Where reactions such as substitutions give a product mixture, thedesired intermediate is optionally separated from other products or atleast partially enriched (e.g., enriched at least about 10-fold, usuallyat least about 50-100-fold) from other products before subsequentreactions are conducted. Substitution at steroid carbon atoms willgenerally proceed with greatest efficiency at the 3-position, which isrelatively sterically unhindered and C-17 is generally somewhat lessaccessible than the C-3 position. The relative reactivities of the C-3,C-7, C-17 and C-16 positions allow one to use their reactivities tocontrol the sequential introduction of different functional groups intothe same steroid molecule. Also, groups, such as hydroxyl at morereactive positions, C-3 or C-17, may be sequentially protected ordeprotected to allow introduction of functional groups at otherpositions, such as C-7 or C-16.

Polymers such as PEG are linked to the compounds essentially asdescribed above. For example, PEG200 or PEG300 is linked to the steroidat the 3, 7, 16, 17 or other positions by an ether linkage(PEG-O-steroid) using a PEG alkoxide (PEG-ONa), to displace the steroidbromide. Alternatively, PEG-Br can be treated with the steroid alkoxide.Polyethylene glycol esters such as those described in U.S. Pat. No.5,681,964 can also be prepared using a suitable formula 1 compound andthe methods described therein. Monosaccharides or polysaccharides andoligonucleotides are linked to steroid hydroxyl groups using knownmethods, see e.g., U.S. Pat. No. 5,627,270.

Formula 1 steroid analogs that comprise one or more ring heteroatoms aresynthesized according to the following methods.

Scheme 11. Formula 1 compounds that comprise two or three ringheteroatoms are prepared as shown in the schemes. In the scheme, X is—CH₂—, —NH—, —O—, or —S—; R⁴⁰ is —H or Br; R⁴¹ is an organic moietyhaving about 12 carbon atoms or less, typically C1-C8 optionallysubstituted alkyl (e.g., methyl, hydroxymethyl, ethyl, propyl) or C2-C8optionally substituted alkenyl having a single double bond (e.g., vinyl)with 1, 2, 3 or more independently selected substituents (e.g., —OH,—COOH, —O—) and with any substituents that comprise a functional groupgenerally being protected. Preparation of compound 20 from 19 isaccomplished using a glycol such as HOC(CH₃)₂C(CH₃)₂OH in acid (H⁺) (B.H. Lipshutz et al., Synth. Commun. 12: 267, 1982). The use of a bulkyprotecting group facilitates generation of a double bond at the 5-6position over the 4-5 position.

Schemes 12A-12D. Compounds of structure 12 are generated as shown in theschemes below. Most of the reactions are conducted essentially asdescribed. See e.g., W. D. Langley, Org. Syn. I, 122, 1932 (compound30); R. Ratcliffe et al., J. Org. Chem. 35: 4000, 1970 (compound 32); A.I. Meyers et al., J. Org. Chem. 39: 2787, 1974 (compound 33, 41); J. L.Isidor et al., J. Org. Chem. 38: 544, 1973 (compound 35); G. Wittig etal., Chem. Ber. 87: 1318, 1954 (compound 36); P. M. Pojer et al., LettLeff. 3067, 1976 (compound 38); A. Maercker, Org. React. 14: 270, 1965(compound 37); E. J. Corey et al., Tet Lett. 3269 1975 (compound 37); R.S. Tipson, J. Org. Chem. 9: 235, 1944 (compound 39); G. W. Kabalka, J.Org. Chem. 51: 2386, 1986; B. B. Carson et al., Org. Synth. 1: 179, 1941(compound 43); H. J. Bestman et al., Justus Liebigs Ann. Chem. 693: 1321966 (compound 39); M. Miyano et al., J. Org. Chem. 37: 268, 1972(compound 51); W. H. Glaze et al., J. Org. Chem. 33: 1987, 1968(compound 52).

Compounds of structure 12 where X is NH, S and CH₂ are prepared as shownin schemes 12B, 12C and 12D respectively.

Scheme 13. The scheme and reactions shown below are used to prepare thecompound of structure 13 and related compounds that are used tointroduce oxygen, carbon, nitrogen or sulfur into the R⁷ and R⁸positions of formula 1 compounds. The reactant in the preparation ofcompound 63, 3-chloro-2-methylpropene (reg. No. 563-47-3), is availablecommercially (e.g., Aldrich, Fluka).

Compound 59 and analogs of compound 59 where CH₂, S or NH CH₂ replacesoxygen are prepared as shown in the following reactions. Conditionssuitable for conversion of compound 106 to 107 have been described (T.Hamada et al., Heterocycles 12: 647, 1979; T. Hamada et al., J. Am.Chem. Soc. 108: 140, 1986).

Conversion of the methyl ketone (—C(O)—CH₃) moiety in compounds havingthe structure

(R—C(O)—CH₃) to other functionalities is accomplished as follows. Themethyl ketone is cleaved to yield a carboxyl moiety using, e.g., Br₂ orI₂ in base, followed by treatment with acid (H₃O⁺) essentially asdescribed (S. J. Chakrabarty Oxidations in Organic Chemistry Part C, W.Trahnnousy, editor, Academic Press, NY, 1987, chapter 5; L. J. Smith etal., Org. Synth. III 302, 1953), to yield R—C(O)—OH. The carboxylic acidis reduced to the alcohol using LiAlH₄. Conversion of the carboxylicacid to the bromide is accomplished using, e.g., Br₂ in water,essentially as described (J. S. Meck et al., Org. Synth. V, 126, 1973;A. Mckillop et al., J. Org. Chem. 34: 1172, 1969).

Compounds of structure 11 are brominated using N-bromosuccinimide toobtain steroids and analogs with bromine at the 7-position.

The 11A compounds are deprotected to yield the aldehyde compounds 12. Asshown in scheme 11, the bromine atom is ultimately found at the7-position. The bromine may be converted to a hydroxyl by reaction ofthe steroid with base (e.g., aqueous KOH), and the hydroxyl may in turnbe protected using known methods, e.g., using C₆H₅—CH₂—Br and base(KOH). The alcohol is protected and deprotected essentially usingdescribed methods, see, e.g., W. H. Hartung et al., Org. React. 7: 263,1953; E. J. Rerst et al., J. Org. Chem. 29: 3725, 1968; A. M. Felix etal., J. Org. Chem. 43: 4194, 1978; D. A. Evans et al., J. Am. Chem. Soc.101: 6789, 1979; international publication number WO 98/02450. Similarreactions are used to convert a bromine at other positions to ahydroxyl. Other substituents are linked to the steroids as described inschemes 1-10.

Alternative routes to introduce a functional group into the 7-positionare also suitable. For example, formula 1 compounds that have a doublebond at the 5-6 position and are unsubstituted at the 7-position areoptionally protected, e.g., hydroxyl groups are protected with acetate,and a ketone is introduced into the 7-position by oxidation with chromicacid essentially as described (U.S. Pat. No. 2,170,124). The carbonyl(═O) at 7 is reduced to a hydroxyl using mild conditions, e.g.,Al(Oi-Pr)₃, to avoid reducing the 5-6 double bond. The use of strongerreducing conditions, e.g., reduction with LiBH₄ in THF, leads toconversion of the 7-carbonyl to hydroxyl and to reduction of the 5-6double bond and other double bonds that may be present in the molecule.

Selective hydrogenation of a double bond at the 16-17 position withoutreduction of a double bond at 5-6 is accomplished using H₂ and Pd. Ingeneral, ketones (═O) can be protected using a glycol, e.g., reactionwith ethylene glycol in p-toluenesulfonic acid and benzene, beforesubsequent oxidation or reduction reactions are conducted.

Various groups that may comprise the formula 1 compounds describedherein, e.g., hydroxyl groups or ketones bonded to the steroid nucleus,or substituted alkyl groups, substituted heterocycles, amino acids andpeptides, can contain one or more reactive moieties such as hydroxyl,carboxyl, amino or thiol. Intermediates used to make formula 1 compoundsmay be protected as is apparent in the art. Noncyclic and cyclicprotecting groups and corresponding cleavage reactions are described in“Protective Groups in Organic Chemistry”, Theodora W. Greene (John Wiley& Sons, Inc., New York, 1991, ISBN 0-471-62301-6) (hereafter “Greene”)and will not be detailed here. In the context of the present invention,these protecting groups are groups that can be removed from the moleculeof the invention without irreversibly changing the covalent bondstructure or oxidation/reduction state of the remainder of the molecule.For example, the protecting group, —R^(PR), that is bonded to an —O— or—NH— group can be removed to form —OH or —NH₂, respectively, withoutaffecting other covalent bonds in the molecule. At times, when desired,more than one protecting group can be removed at a time, or they can beremoved sequentially. In compounds of the invention containing more thanone protecting group, the protecting groups are the same or different.

Protecting groups are removed by known procedures, although it will beunderstood that the protected intermediates fall within the scope ofthis invention. The removal of the protecting group may be arduous orstraightforward, depending upon the economics and nature of theconversions involved. In general, one will use a protecting group withexocyclic amines or with carboxyl groups during synthesis of a formula 1compound. For most therapeutic applications amine groups should bedeprotected. Protecting groups commonly are employed to protect againstcovalent modification of a sensitive group in reactions such asalkylation or acylation. Ordinarily, protecting groups are removed by,e.g. hydrolysis, elimination or aminolysis. Thus, simple functionalconsiderations will suffice to guide the selection of a reversible or anirreversible protecting group at a given locus on the inventioncompounds. Suitable protecting groups and criteria for their selectionare described in T. W. Greene and P. G. M. Wuts, Eds. “Protective Groupsin Organic Synthesis” 2nd edition, Wiley Press, at pps. 10-142, 143-174,175-223, 224-276, 277-308, 309-405 and 406-454.

Determination of whether a group is a protecting group is made in theconventional manner, e.g., as illustrated by Kocienski, Philip J.;“Protecting Groups” (Georg Thieme Verlag Stuttgart, New York, 1994)(hereafter “Kocienski”), Section 1.1, page 2, and Greene Chapter 1,pages 1-9. In particular, a group is a protecting group if when, basedon mole ratio, 90% of that protecting group has been removed by adeprotection reaction, no more than 50%, typically 25%, more typically10%, of the deprotected product molecules of the invention haveundergone changes to their covalent bond structure oroxidation/reduction state other than those occasioned by the removal ofthe protecting group. When multiple protecting groups of the same typeare present in the molecule, the mole ratios are determined when all ofthe groups of that type are removed. When multiple protecting groups ofdifferent types are present in the molecule, each type of protectinggroup is treated (and the mole ratios are determined) independently ortogether with others depending on whether the deprotection reactionconditions pertinent to one type are also pertinent to the other typespresent. In one embodiment of the invention, a group is a protectinggroup if when, based on mole ratio determined by conventionaltechniques, 90% of that protecting group has been removed by aconventional deprotection reaction, no more than 50%, typically 25%,more typically 10%, of the deprotected product molecules of theinvention have undergone irreversible changes to their covalent bondstructure or oxidation/reduction state other than those occasioned bythe removal of the protecting group. Irreversible changes requirechemical reactions (beyond those resulting from aqueous hydrolysis,acid/base neutralization or conventional separation, isolation orpurification) to restore the covalent bond structure oroxidation/reduction state of the deprotected molecule of the invention.

Protecting groups are also described in detail together with generalconcepts and specific strategies for their use in Kocienski, Philip J.;“Protecting Groups” (Georg Thieme Verlag Stuttgart, New York, 1994),which is incorporated by reference in its entirety herein. In particularChapter 1, Protecting Groups: An Overview, pages 1-20, Chapter 2,Hydroxyl Protecting Groups, pages 21-94, Chapter 3, Diol ProtectingGroups, pages 95-117, Chapter 4, Carboxyl Protecting Groups, pages118-154, Chapter 5, Carbonyl Protecting Groups, pages 155-184, Chapter6, Amino Protecting Groups, pages 185-243, Chapter 7, Epilog, pages244-252, and Index, pages 253-260, are incorporated with specificity inthe context of their contents. More particularly, Sections 2.3 SilylEthers, 2.4 Alkyl Ethers, 2.5 Alkoxyalkyl Ethers (Acetals), 2.6 Reviews(hydroxy and thiol protecting groups), 3.2 Acetals, 3.3 SilyleneDerivatives, 3.4 1,1,3,3-Tetraisopropyldisiloxanylidene Derivatives, 3.5Reviews (diol protecting groups), 4.2 Esters, 4.32,6,7-Trioxabicyclo[2.2.2]octanes [OBO] and Other Ortho Esters, 4.4Oxazolines, 4.5 Reviews (carboxyl protecting groups), 5.2 O,O-Acetals,5.3 S,S-Acetals, 5.4 O,S-Acetals, 5.5 Reviews (carbonyl protectinggroups), 6.2 N-Acyl Derivatives, 6.3 N-Sulfonyl Derivatives, 6.4N-Sulfenyl Derivatives, 6.5 N-Alkyl Derivatives, 6.6 N-SilylDerivatives, 6.7 Imine Derivatives, and 6.8 Reviews (amino protectinggroups), are each incorporated with specificity whereprotection/deprotection of the requisite functionalities is discussed.Further still, the tables “Index to the Principal Protecting Groups”appearing on the inside front cover and facing page, “Abbreviations” atpage xiv, and “reagents and Solvents” at page xv are each incorporatedin their entirety herein at this location.

Typical hydroxy protecting groups are described in Greene at pages14-118 and include Ethers (Methyl); Substituted Methyl Ethers(Methoxymethyl, Methylthiomethyl, t-Butylthiomethyl,(Phenyldimethylsilyl)methoxymethyl, Benzyloxymethyl,p-Methoxybenzyloxymethyl, (4-Methoxyphenoxy)methyl, Guaiacolmethyl,t-Butoxymethyl, 4-Pentenyloxymethyl, Siloxymethyl,2-Methoxyethoxymethyl, 2,2,2-Trichloroethoxymethyl,Bis(2-chloroethoxy)methyl, 2-(Trimethylsilyl)ethoxymethyl,Tetrahydropyranyl, 3-Bromotetrahydropyranyl, Tetrahydropthiopyranyl,1-Methoxycyclohexyl, 4-methoxytetrahydropyranyl,4-Methoxytetrahydrothiopyranyl, 4-MethoxytetrahydropthiopyranylS,S-Dioxido, 1-[(2-Chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl,1,4-Dioxan-2-yl, Tetrahydrofuranyl, Tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-Octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl);Substituted Ethyl Ethers (1-Ethoxyethyl, 1-(2-Chloroethoxy)ethyl,1-Methyl-1-methoxyethyl, 1-Methyl-1-benzyloxyethyl,1-Methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-Trichloroethyl,2-Trimethylsilylethyl, 2-(Phenylselenyl)ethyl, t-Butyl, Allyl,p-Chlorophenyl, p-Methoxyphenyl, 2,4-Dinitrophenyl, Benzyl); SubstitutedBenzyl Ethers (p-Methoxybenzyl, 3,4-Dimethoxybenzyl, o-Nitrobenzyl,p-Nitrobenzyl, p-Halobenzyl, 2,6-Dichlorobenzyl, p-Cyanobenzyl,p-Phenylbenzyl, 2- and 4-Picolyl, 3-Methyl-2-picolyl N-Oxido,Diphenylmethyl, p, p′-Dinitrobenzhydryl, 5-Dibenzosuberyl,Triphenylmethyl, alpha-Naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, Di(p-methoxyphenyl)phenylmethyl,Tri(p-methoxyphenyl)methyl, 4-(4′Bromophenacyloxy)phenyldiphenylmethyl,4,4′,4″-Tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-Tris(levulinoyloxyphenyl)methyl,4,4′,4″-Tris(benzoyloxyphenyl)methyl,3-(Imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-Bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-Anthryl,9-(9-Phenyl)xanthenyl, 9-(9-Phenyl-10-oxo)anthryl,1,3-Benzodithiolan-2-yl, Benzisothiazolyl, S,S-Dioxido); Silyl Ethers(Trimethylsilyl, Triethylsilyl, Triisopropylsilyl,Dimethylisopropylsilyl, Diethylisopropylsily, Dimethylthexylsilyl,t-Butyldimethylsilyl, t-Butyldiphenylsilyl, Tribenzylsilyl,Tri-p-xylylsilyl, Triphenylsilyl, Diphenylmethylsilyl,t-Butylmethoxyphenylsilyl); Esters (Formate, Benzoylformate, Acetate,Choroacetate, Dichloroacetate, Trichloroacetate, Trifluoroacetate,Methoxyacetate, Triphenyl-methoxyacetate, Phenoxyacetate,p-Chlorophenoxyacetate, p-poly-Phenylacetate, 3-Phenylpropionate,4-Oxopentanoate (Levulinate), 4,4-(Ethylenedithio)pentanoate, Pivaloate,Adamantoate, Crotonate, 4-Methoxycrotonate, Benzoate, p-Phenylbenzoate,2,4,6-Trimethylbenzoate (Mesitoate); Carbonates (Methyl,9-Fluorenylmethyl, Ethyl, 2,2,2-Trichloroethyl, 2-(Trimethylsilyl)ethyl,2-(Phenylsulfonyl)ethyl, 2-(Triphenylphosphonio)ethyl, Isobutyl, Vinyl,Allyl, p-Nitrophenyl, Benzyl, p-Methoxybenzyl, 3,4-Dimethoxybenzyl,o-Nitrobenzyl, p-Nitrobenzyl, S-Benzyl Thiocarbonate,4-Ethoxy-1-naphthyl, Methyl Dithiocarbonate); Groups With AssistedCleavage (2-Iodobenzoate, 4-Azidobutyrate, 4-Nitro-4-methylpentanoate,o-(Dibromomethyl)benzoate, 2-Formylbenzenesulfonate,2-(Methylthiomethoxy)ethyl Carbonate, 4-(Methylthiomethoxy)butyrate,2-(Methylthiomethoxymethyl)benzoate); Miscellaneous Esters(2,6-Dichloro-4-methylphenoxyacetate,2,6-Dichloro-4-(1,1,3,3-tetramethyl-butyl)phenoxyacetate,2,4-Bis(1,1-dimethylpropyl)phenoxyacetate, Chorodiphenylacetate,Isobutyrate, Monosuccinoate, (E)-2-Methyl-2-butenoate (Tigloate),o-(Methoxycarbonyl)benzoate, p-poly-Benzoate, α-Naphthoate, Nitrate,Alkyl N,N,N′,N′-Tetramethylphosphorodiamidate, N-Phenylcarbamate,Borate, Dimethylphosphinothioyl, 2,4-Dinitro-phenylsulfenate); andSulfonates (Sulfate, Methanesulfonate (Mesylate), Benzylsulfonate,Tosylate (Tos)).

More typically hydroxy protecting groups include substituted methylethers, substituted benzyl ethers, silyl ethers, and esters includingsulfonic acid esters, still more typically, trialkylsilyl ethers,tosylates and acetates.

Typical 1,2- and 1,3-diol protecting groups are described in Greene atpages 118-142 and include Cyclic Acetals and Ketals (Methylene,Ethylidene, 1-t-Butylethylidene, 1-Phenylethylidene,(4-Methoxyphenyl)ethylidene, 2,2,2-Trichloroethylidene, Acetonide(Isopropylidene), Cyclopentylidene, Cyclohexylidene, Cycloheptylidene,Benzylidene, p-Methoxybenzylidene, 2,4-Dimethoxybenzylidene,3,4-Dimethoxybenzylidene, 2-Nitrobenzylidene); Cyclic Ortho Esters(Methoxymethylene, Ethoxymethylene, Dimethoxymethylene,1-Methoxyethylidene, 1-Ethoxyethylidine, 1,2-Dimethoxyethylidene,alpha-Methoxybenzylidene, 1-(N,N-Dimethylamino)ethylidene Derivative,alpha-(N,N-Dimethylamino)benzylidene Derivative, 2-Oxacyclopentylidene);and Silyl Derivatives (Di-t-butylsilylene Group,1,3-(1,1,3,3-Tetraiso-propyldisiloxanylidene) Derivative,Tetra-t-butoxydisiloxane-1,3-diylidene Derivative, Cyclic Carbonates,Cyclic Boronates, Ethyl Boronate, Phenyl Boronate).

More typically, 1,2- and 1,3-diol protecting groups include epoxides andacetonides.

Typical amino protecting groups are described in Greene at pages 315-385and include Carbamates (Methyl and Ethyl, 9-Fluorenylmethyl,9(2-Sulfo)fluoroenylmethyl, 9-(2,7-Dibromo)fluorenylmethyl,2,7-Di-t-buthyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]-methyl,4-Methoxy-phenacyl); Substituted Ethyl (2,2,2-Trichoroethyl,2-Trimethylsilylethyl, 2-Phenylethyl, 1-(1-Adamantyl)-1-methylethyl,1,1-Dimethyl-2-haloethyl, 1,1-Dimethyl-2,2-dibromoethyl,1,1-Dimethyl-2,2,2-trichloroethyl, 1-Methyl-1-(4-biphenylyl)ethyl,1-(3,5-Di-t-butylphenyl)-1-methylethyl, 2-(2′- and 4′-Pyridyl)ethyl,2-(N,N-Dicyclohexylcarboxamido)ethyl, t-Butyl, 1-Adamantyl, Vinyl,Allyl, 1-Isopropylallyl, Cinnamyl, 4-Nitrocinnamyl, 8-Quinolyl,N-Hydroxypiperidinyl, Alkyldithio, Benzyl, p-Methoxybenzyl,p-Nitrobenzyl, p-Bromobenzyl, p-Chorobenzyl, 2,4-Dichlorobenzyl,4-Methylsulfinylbenzyl, 9-Anthrylmethyl, Diphenylmethyl); Groups WithAssisted Cleavage (2-Methylthioethyl, 2-Methylsulfonylethyl,2-(p-Toluenesulfonyl)ethyl, [2-(1,3-Dithianyl)]methyl,4-Methylthiophenyl, 2,4-Dimethylthiophenyl, 2-Phosphonioethyl,2-Triphenylphosphonioisopropyl, 1,1-Dimethyl-2-cyanoethyl,m-Choro-p-acyloxybenzyl, p-(Dihydroxyboryl)benzyl,5-Benzisoxazolylmethyl, 2-(Trifluoromethyl)-6-chromonylmethyl); GroupsCapable of Photolytic Cleavage (m-Nitrophenyl, 3,5-Dimethoxybenzyl,o-Nitrobenzyl, 3,4-Dimethoxy-6-nitrobenzyl,Phenyl(o-nitrophenyl)methyl); Urea-Type Derivatives(Phenothiazinyl-(10)-carbonyl Derivative,N′-p-Toluenesulfonylaminocarbonyl, N′-Phenylaminothiocarbonyl);Miscellaneous Carbamates (t-Amyl, S-Benzyl Thiocarbamate, p-Cyanobenzyl,Cyclobutyl, Cyclohexyl, Cyclopentyl, Cyclopropylmethyl,p-Decyloxybenzyl, Diisopropylmethyl, 2,2-Dimethoxycarbonylvinyl,o-(N,N-Dimethyl-carboxamido)benzyl,1,1-Dimethyl-3-(N,N-dimethylcarboxamido)propyl, 1,1-Dimethylpropynyl,Di(2-pyridyl)methyl, 2-Furanylmethyl, 2-Iodoethyl, Isobornyl, Isobutyl,Isonicotinyl, p-(p′-Methoxyphenylazo)benzyl, 1-Methylcyclobutyl,1-Methylcyclohexyl, 1-Methyl-1-cyclopropylmethyl,1-Methyl-1-(3,5-dimethoxyphenyl)ethyl,1-Methyl-1-(p-phenylazophenyl)ethyl, 1-Methyl-1-phenylethyl,1-Methyl-1-(4-pyridyl)ethyl, Phenyl, p-(Phenylazo)-benzyl,2,4,6-Tri-t-butylphenyl, 4-(Trimethylammonium)benzyl,2,4,6-Trimethylbenzyl); Amides (N-Formyl, N-Acetyl, N-Choroacetyl,N-Trichoroacetyl, N-Trifluoroacetyl, N-Phenylacetyl,N-3-Phenylpropionyl, N-Picolinoyl, N-3-Pyridylcarboxamide,N-Benzoylphenylalanyl Derivative, N-Benzoyl, N-p-Phenylbenzoyl); AmidesWith Assisted Cleavage (N-o-Nitrophenylacetyl, N-o-Nitrophenoxyacetyl,N-Acetoacetyl, (N′-Dithiobenzyloxycarbonylamino)acetyl,N-3-(p-Hydroxyphenyl)propionyl, N-3-(o-Nitrophenyl)propionyl,N-2-Methyl-2-(o-nitrophenoxy)propionyl,N-2-Methyl-2-(o-phenylazophenoxy)propionyl, N-4-Chlorobutyryl,N-3-Methyl-3-nitrobutyryl, N-o-Nitrocinnamoyl, N-AcetylmethionineDerivative, N-o-Nitrobenzoyl, N-o-(Benzoyloxymethyl)benzoyl,4,5-Diphenyl-3-oxazolin-2-one); Cyclic Imide Derivatives (N-Phthalimide,N-Dithiasuccinoyl, N-2,3-Diphenylmaleoyl, N-2,5-Dimethylpyrrolyl,N-1,1,4,4-Tetramethyl-disilylazacyclopentane Adduct, 5-Substituted1,3-Dimethyl-1,3,5-triazacyclo-hexan-2-one, 5-Substituted1,3-Dibenzyl-1,3,5-triazacyclohexan-2-one, 1-Substituted3,5-Dinitro-4-pyridonyl); N-Alkyl and N-Aryl Amines (N-Methyl, N-Allyl,N-[2-(Trimethylsilyl)ethoxy]methyl, N-3-Acetoxypropyl,N-(1-Isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl), Quaternary AmmoniumSalts, N-Benzyl, N-Di(4-methoxyphenyl)methyl, N-5-Dibenzosuberyl,N-Triphenylmethyl, N-(4-Methoxyphenyl)diphenylmethyl,N-9-Phenylfluorenyl, N-2,7-Dichloro-9-fluorenylmethylene,N-Ferrocenylmethyl, N-2-Picolylamine N′-Oxide); Imine Derivatives(N-1,1-Dimethylthiomethylene, N-Benzylidene, N-p-methoxybenylidene,N-Diphenylmethylene, N-[(2-Pyridyl)mesityl]methylene,N,(N′,N′-Dimethylaminomethylene, N,N′-Isopropylidene,N-p-Nitrobenzylidene, N-Salicylidene, N-5-Chlorosalicylidene,N-(5-Chloro-2-hydroxyphenyl)phenylmethylene, N-Cyclohexylidene); EnamineDerivative (N-(5,5-Dimethyl-3-oxo-1-cyclohexenyl)); N-Metal Derivatives(N-Borane Derivatives, N-Diphenylborinic Acid Derivative,N-[Phenyl(pentacarbonylchromium- or -tungsten)]carbenyl, N-Copper orN-Zinc Chelate); N-N Derivatives (N-Nitro, N-Nitroso, N-Oxide); N-PDerivatives (N-Diphenylphosphinyl, N-Dimethylthiophosphinyl,N-Diphenylthiophosphinyl, N-Dialkyl Phosphoryl, N-Dibenzyl Phosphoryl,N-Diphenyl Phosphoryl); N-Si Derivatives; N-S Derivatives; N-SulfenylDerivatives (N-Benzenesulfenyl, N-o-Nitrobenzenesulfenyl,N-2,4-Dinitrobenzenesulfenyl, N-Pentachlorobenzenesulfenyl,N-2-nitro-4-methoxybenzenesulfenyl, N-Triphenylmethylsulfenyl,N-3-Nitropyridinesulfenyl); and N-Sulfonyl Derivatives(N-p-Toluenesulfonyl, N-Benzenesulfonyl,N-2,3,6-Trimethyl-4-methoxybenzenesulfonyl,N-2,4,6-Trimethoxybenzenesulfonyl,N-2,6-Dimethyl-4-methoxybenzenesulfonyl, N-Pentamethylbenzenesulfonyl,N-2,3,5,6-Tetramethyl-4-methoxybenzenesulfonyl,N-4-methoxybenzenesulfonyl, N-2,4,6-Trimethylbenzenesulfonyl,N-2,6-Dimethoxy-4-methylbenzenesulfonyl,N-2,2,5,7,8-Pentamethylchroman-6-sulfonyl, N-Methanesulfonyl,N-.beta.-Trimethylsilyethanesulfonyl, N-9-Anthracenesulfonyl,N-4-(4′,8′-Dimethoxynaphthylmethyl)benzenesulfonyl, N-Benzylsulfonyl,N-Trifluoromethylsulfonyl, N-Phenacylsulfonyl).

More typically, amino protecting groups include carbamates and amides,still more typically, N-acetyl groups.

Groups capable of biological cleavage typically include prodrugs. Alarge number of such groups are described in “Design of Prodrugs”, HansBundgaard (Elsevier, N.Y., 1985, ISBN 0-444-80675-X) (Bundgaard) andwill not be detailed here. In particular, Bundgaard, at pages 1-92,describes prodrugs and their biological cleavage reactions for a numberof functional group types. Prodrugs for carboxyl and hydroxyl groups aredetailed in Bundgaard at pages 3 to 10, for amides, imides and otherNH-acidic compounds at pages 10 to 27, amines at pages 27 to 43, andcyclic prodrugs at pages 62 to 70. These moieties are optionally bondedto the steroid at one two or more of R¹-R⁶, R¹⁰, R¹⁵, R¹⁷ and R¹⁸.

Metabolites. Also falling within the scope of this invention are the invivo metabolites of the compounds described herein, to the extent suchproducts are novel and unobvious over the prior art. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,esterification and the like of the administered formula 1 compound, dueto enzymatic or chemical processes. Accordingly, the invention includesnovel and unobvious compounds produced by a process comprisingcontacting a compound of this invention with a subject, e.g., a human,rodent or a primate, for a period of time sufficient to yield ametabolic product thereof. Such products typically are identified bypreparing a radiolabeled (e.g. ¹⁴C, ³H, ¹³¹I, ³²P, ³⁵S or ⁹⁹Tc) compoundof the invention, administering it parenterally in a detectable dose(e.g. greater than about 0.5 mg/kg) to an animal such as rat, mouse,guinea pig, primate, or to a human, allowing sufficient time formetabolism to occur (typically about 30 seconds to 30 hours) andisolating its conversion products from the urine, blood or otherbiological samples. These products are easily isolated since they arelabeled (others are isolated by the use of antibodies capable of bindingepitopes surviving in the metabolite). The metabolite structures aredetermined in conventional fashion, e.g. by MS, HPLC or NMR analysis. Ingeneral, analysis of metabolites is done in the same way as conventionaldrug metabolism studies well-known to those skilled in the art. Theconversion products, so long as they are not otherwise found in vivo,are useful in diagnostic assays for therapeutic dosing of the compoundsof the invention even if they possess no therapeutic activity of theirown.

Formulations and compositions for preparing formulations. While it ispossible for the active ingredient(s) to be administered alone it isusual to present them as pharmaceutical formulations. The formulations,both for veterinary and for human use, of the invention comprise atleast one active ingredient, i.e., a formula 1 compound, together withone or more acceptable excipients therefor and optionally othertherapeutic ingredients.

Another aspect of the invention relates to compositions comprising oneor more pharmaceutically acceptable excipients or carriers. One or moreformula 1 compound(s) (also referred to as the “active ingredient(s)”)are administered by any route appropriate to the condition to betreated. Suitable routes for the non-aqueous liquid formulations andother formula 1 compound formulations include oral, rectal, nasal,topical (including buccal and sublingual), vaginal and parenteral(including subcutaneous, intramuscular, intravenous, intradermal,intrathecal and epidural). In general, the non-aqueous liquidformulations are delivered by a parenteral route. In other embodiments,such as the invention intermittent dosing methods, the formula 1compound(s) may be present as a non-aqueous liquid formulation, a drysolid formulation that is an oral, topical, parenteral formulation, oras an aqueous liquid formulation that is used parenterally, orally ortopically. It will be appreciated that the preferred route may varywith, for example, the subject's pathological condition or weight or thesubject's response to therapy with a formula 1 compound or other therapyappropriate to the circumstances.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques, excipients and formulations generally are foundin, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa. 1985, 17^(th) edition, Nema et al., PDA J. Pharm. Sci. Tech.1997 51:166-171. Methods to make invention formulations include the stepof bringing into association an active ingredient(s) with theexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidexcipients or finely divided solid excipients or both, and then, ifappropriate, shaping the product.

Formulations of the invention suitable for oral administration areprepared as discrete units such as capsules, cachets or tablets eachcontaining a predetermined amount of the active ingredient; as a powderor granules; as solution or a suspension in an aqueous liquid or anon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient(s) may also bepresented as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient(s) in a free-flowing formsuch as a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient(s) moistened with an inert liquid diluent.The tablets may optionally be coated or scored and optionally areformulated so as to provide slow or controlled release of the activeingredient(s) therefrom.

For infections of the eye or other external tissues e.g. mouth and skin,the formulations are typically applied as a topical ointment or creamcontaining the active ingredient(s) in an amount of, for example, 0.075to 20% w/w (including active ingredient(s) in a range between 0.1% and20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.), often0.2 to 15% w/w and most often 0.5 to 10% w/w. When formulated in anointment, the active ingredients may be employed with either aparaffinic or a water-miscible ointment base. Alternatively, the activeingredients may be formulated in a cream with an oil-in-water creambase.

If desired, the aqueous phase of the cream base may include, forexample, at least 30% w/w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG 400) and mixtures thereof. The topical formulations maydesirably include a compound that enhances absorption or penetration ofthe active ingredient(s) through the skin or other affected areas.Examples of such dermal penetration enhancers include dimethylsulphoxide and related analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known excipients in a known manner. While the phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. A hydrophilic emulsifier may be includedtogether with a lipophilic emulsifier, which acts as a stabilizer. Someembodiments include both an oil and a fat. Together, the emulsifier(s)with or without stabilizer(s) make up the so-called emulsifying wax, andthe wax together with the oil and fat make up the so-called emulsifyingointment base which forms the oily dispersed phase of the creamformulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the invention include TWEEN60™, SPAN60™, cetostearyl alcohol, benzylalchol, myristy alcohol, glyceryl mono-stearate and sodium laurylsulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties. Creams are generally anon-greasy, non-staining and washable products with suitable consistencyto avoid leakage from tubes or other containers. Straight or branchedchain, mono- or dibasic alkyl esters such as di-isoadipate, isocetylstearate, propylene glycol diester of coconut fatty acids, isopropylmyristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used. These may be used alone or in combinationdepending on the properties required. Alternatively, high melting pointlipids such as white soft paraffin and/or liquid paraffin or othermineral oils are used.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient(s) is dissolved or suspended ina suitable excipient(s), especially an aqueous solvent for activeingredient(s) that comprise one or more charges at pH values nearneutrality, e.g., about pH 6-8. The active ingredient(s) is typicallypresent in such formulations in a concentration of about 0.5-20% w/w,typically about 1-10% w/w, often about 2-5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient(s) in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid excipient(s).

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.01 to 500 microns (includingaverage particle sizes in a range between 0.01 and 500 microns in 0.1micron or other increments, e.g., 0.05, 0.1, 0.5, 1, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 20, 25, 30, 35, 50, 75, 100, etc.microns), which is administered by rapid inhalation through the nasalpassage or by inhalation through the mouth so as to reach the alveolarsacs. Suitable micronized formulations include aqueous or oily solutionsor suspensions of the active ingredient(s). Formulations suitable foraerosol, dry powder or tablet administration may be prepared accordingto conventional methods and may be delivered with other therapeuticagents such as compounds heretofore used in the treatment or prophylaxisof viral or other infections as described herein. Such formulation maybe administered, e.g., orally, parenterally (i.v., i.m., s.c.),topically or by a buccal route.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient(s) such excipients asare known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

The formulations are presented in unit-dose or multi-dose containers,for example sealed ampules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Unit dosage formulations are those containing adaily dose or unit daily sub-dose, as recited herein, or an appropriatefraction thereof, of the active ingredient(s).

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents or excipients conventional in the art having regard to the typeof formulation in question, for example those suitable for oraladministration may include flavoring agents.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinaryexcipient(s) therefor. Veterinary excipients are materials useful forthe purpose of administering the composition and may be solid, liquid orgaseous materials that are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient(s). Theseveterinary compositions may be administered orally, parenterally or byany other desired route.

Invention formulations include controlled release pharmaceuticalformulations containing an active ingredient(s) (“controlled releaseformulations”) in which the release of the active ingredient(s) iscontrolled and regulated to allow less frequency dosing or to improvethe pharmacokinetic or toxicity profile of a given active ingredient(s).

An effective dose of active ingredient(s) depends at least on the natureof the condition being treated, toxicity, whether the compound(s) isbeing used prophylactically (lower doses) or against an active infectionor condition, the method of delivery, and the pharmaceuticalformulation, and will be determined by the clinician using conventionaldose escalation studies. It can be expected to be from about 0.05 toabout 30 mg/kg body weight per day. For example, for topical deliverythe daily candidate dose for an adult human of approximately 70 kg bodyweight will range from about 1 mg to about 500 mg, generally betweenabout 5 mg and about 40 mg, and may take the form of single or multipledoses or administration sites.

Embodiments include formulations that comprise a liposome or lipidcomplex that comprises a formula 1 compound(s), e.g., BrEA or an ester,carbamate, carbonate, amino acid or peptide thereof. Such formulationsare prepared according to known methods, e.g., U.S. Pat. Nos. 4,427,649,5,043,165, 5,714,163, 5,744,158, 5,783,211, 5,795,589, 5,795,987,5,798,348, 5,811,118, 5,820,848, 5,834,016 and 5,882,678. The liposomesoptionally contain an additional therapeutic agent(s), e.g.,amphotericin B, cis-platin, adriamycin, a protease inhibitor, anucleoside or a nucleotide analog, such as one of those mentionedherein. Formulations that comprise liposomes can be delivered to asubject by any standard route, e.g., oral, aerosol or parenteral (e.g.,s.c., i.v. or i.m.).

Therapeutic applications. The formula 1 compounds, or the biologicallyactive substances produced from these compounds by hydrolysis ormetabolism in vivo, have a number of clinical and non-clinicalapplications. The compounds are generally useful to enhance Th1 immuneresponses or to reduce Th2 immune responses. As used herein, referenceto Th1 or Th2 immune responses means such responses as observed inmammals generally and not as observed in the murine system, from whichthe Th1 and Th2 terminology originated. Thus, in humans, Th1 cellspreferentially display chemokine receptors CXCR3 and CCR5, while Th2cells preferentially express the CCR4 molecule and a smaller amount ofthe CCR3 molecule.

Aspects of the invention include compositions that comprise an amount ofat least one formula 1 compound effective to enhance the relativeproportion of a desired immune cell subset, e.g., CD4⁺ T cells, NK cellsor dendritic cells, or to modulate one or more functions of immune cellsubsets and a pharmaceutically acceptable carrier. Typical immunemodulation centers on modulating expression of gene(s) that enhance ofTh1 immune responses or reduces of Th2 immune responses. Functions thatthe formula 1 compounds affected include expression of CD molecules oralteration of the proportion of cell subsets, e.g., CD4⁺ or CD8⁺ Tcells, or their relative numbers in a subject's blood or tissues. CDmolecules participate in the function of various immune cell subsets andcan be useful as markers for immune function in vivo. In some aspects,the formula 1 compounds activate immune cells which generally alters(increases or decreases) expression of, or changes the numbers of cellsthat express combinations of, CD4, CD6, CD8, CD25, CD27, CD28, CD30,CD38, CD39, CD43, CD45RA, CD45RO, CD62L, CD69, CD71, CD90 or HLA-DRmolecules. Often, the numbers of cells that express these molecules areincreased, e.g., CD25, CD16 or CD69. Typically such increases areobserved as an increased proportion of circulating white blood cellsthat express one or more of these molecules. In some cases the number ofsuch molecules per cell is detectably altered.

Expression of one or more adhesion molecules CD2, CD5, CD8, CD11a,CD11b, CD11c, CD18, CD29, CD31, CD44, CD49a, CD49b, CD49c, CD49d, CD49e,CD49f, CD50, CD54, CD58, CD103 or CD104 are also detectably affectedafter administration of the formula 1 compounds to a subject. Often, thenumbers of cells that express these molecules are increased, e.g., CD5or CD56. The adhesion molecules function in various aspects of immuneresponses, such as binding to class I MHC molecules, transducing signalsbetween cells or binding to molecules in the extracellular matrixassociated with endothelial or other cell types. Administration of theformula 1 compounds to a subject also affects the numbers of certainimmune cell subsets, e.g., NK cells (e.g., CD8⁻, CD56⁺ or CD8⁺, CD56⁺)or lymphokine activated killer cells (LAK). Increased circulating NK orLAK cells are typically observed, which is reflected in increasednumbers of cells that express one or more of CD16, CD38, CD56, CD57 orCD94. Also, increased numbers of circulating dendritic cell precursorsare observed, as shown by increases in cells that express one or more ofCD11c, CD80, CD83, CD106 or CD123. Although one can observe an increasedproportion of circulating white blood cells that express one or more ofthese molecules, in some instances the number of such molecules per cellis detectably altered. Both the cell numbers and the density of CDmolecule per cell can also be detectably modulated. Modulation of immunecell subsets typically occurs on intermittent dosing of a formula 1compound.

Expression of one or more homing receptors such as CD62L is may also bedetectably affected after administration of the formula 1 compounds to asubject. Often, the numbers of cells that express these molecules areincreased, e.g., CD62L.

Other CD molecules that are modulated by the presence of the formula 1compounds in a subject include cytokine receptor molecules such asCD115, CDW116, CD117, CD118, CDW119, CD120a, CD120b, CD121a, CD121 b,CD122, CD123, CD124, CD125 CD126, CDW127, CDW128 or CDW130. Often, thenumbers of receptor molecules per cell will be modulated. For example,receptors for cytokines that mediate Th1 immune responses (e.g., IL-2,γIFN) will typically increase in or on cells that mediate Th1 immuneresponses. Modulation of these molecules may be by direct interactionswith a receptor(s) in the cell that expresses the cytokine receptor orindirectly by modulation of cytokine synthesis in the affected cells orin other cells, typically immune cells, that may interact with the cellswhose receptor synthesis is being modulated.

Treatment of a subject with a formula 1 compound can result in a changeof at least 25-50% above or below (e.g., at least 30% or at least 40%above or below) the control or basal level of some immune cell subsets.For example, increases of more than about 30% in the total numbers ofactivated CD8⁺ T cells, e.g., CD8⁺, CD69⁺, CD25⁺ T cells, CD8⁺, CD69⁺,CD25⁻ T cells or CD8⁺, CD69⁻, CD25⁺ T cells, usually occurs by 7 daysafter a single dose of a formula 1 compound to a subject. Such increasesmay be greater than 50%, 60% or 100% in the total numbers of activatedCD8⁺ T cells or subsets of activated CD8⁺ T cells in individualsubjects. Typically such increases are about in the total numbers ofactivated CD8⁺ T cells or subsets of activated CD8⁺ T cells averagesabout 30-40%, with individual subjects experiencing increases over 100%in the numbers of activated CD8⁺ T cells per unit blood volume comparedto the basal level.

Administration of the formula 1 compounds can affect other immune cellsubsets. For example, the concentration of circulating CD4⁺, CD69⁺,CD25⁻ (Th1 helper cells) and CD8⁺, CD16⁺, CD38⁺ LAK cells or CD8⁻,CD16⁺, CD38⁺ LAK cells typically increases during or after the course ofdosing a subject with a formula 1 compound. Also, CD8⁻, CD16⁺, CD38⁺ andCD8⁺, CD16⁺, CD38⁺ (ADCC effector cells) and low side scatter Lin⁻, DR⁺,CD123⁺ (dendritic precursors) or low side scatter Lin⁻, DR⁺, CD11c⁺(dendritic cells or precursors) may show modest to significantincreases.

In subjects that are immunosuppressed, e.g., from infection (e.g., viral(HIV, HCV), bacterial infection or parasite infection) or fromchemotherapy (e.g., an antiviral therapy, a cancer chemotherapy or aradiation therapy), administration of the formula 1 compounds to thesubject results in a favorable shift in the balance of Th1 or Th2responses the subject can mount in the face of immunosuppression. WhenTh1 responses are suboptimal or insufficient, treatment with a formula 1compound results in enhancement of Th1 responses or a reduction in Th2responses. Conversely, when Th2 responses are suboptimal orinsufficient, treatment with a formula 1 compound results in enhancementof Th2 responses or a reduction in Th1 responses. The formula 1compounds can thus be used to shift the nature of a subject's immuneresponse to result in a more balanced immune response fromimmunosuppression. Alternatively, the compounds can selectively suppressinappropriate or unwanted immune responses. Enhanced Th1 responsesappears to be at least partly due to one or more of (i) a reduction inbiological restraints, e.g., high levels of IL-4 or IL-10, on Th1functions by preexisting primed Th1 effector cells, (ii) enhanceddifferentiation of Th0 cells to Th1 cells or enhanced responses mediatedby Th1 cells, (iii) enhanced function of accessory cell function, e.g.,antigen presentation by dendritic precursor cells or by macrophages,(iv) enhanced proliferation and differentiation of Th1 precursor orprogenitor cells, (v) enhanced IL-12 expression in dendritic cells ortheir precursors, which results in enhanced differentiation of Th1 cellsfrom Th0 precursors, (vi) enhanced expression or activity of factorsassociated with Th1 functions, e.g., IL-2, gamma interferon (γIFN) orlymphotoxin.

An aspect of the invention methods is an alteration in the expression ofIL-4 or IL-10 that occurs after administration of a formula 1 compound,e.g., BrEA, to a subject. A consistent observation is that extracellularIL-4 or IL-10 levels rapidly decrease to levels that are undetectable byELISA. Intracellular IL-10 levels are reduced to levels that are near orbelow the limits of detection by flow cytometry. The administration of aformula 1 compound to a subject thus provides a means to inhibit eitheror both of these interleukins. Such inhibition may be associated withenhancement of Th1 immune responses relative to Th2 or Th0 responses,e.g., in subjects where Th1 responses are suppressed (e.g., from viral,bacterial or parasite infection (HIV, HCV, etc) or chemotherapy) or areotherwise suboptimal. In many subjects, levels of either IL-4 or IL-10,usually IL-10, before dosing with a formula 1 compound is low orundetectable. In these subjects, dosing with the formula 1 compoundresults in a rapid drop in the interleukin that is detectable, usuallyIL-4.

In some embodiments, the formula 1 compound(s) is administered to asubject who has a pathogen infection, such as a viral, bacterial orparasite infection. The formula 1 compounds can be considered for use ina broad scope of infections (see, e.g., J. B. Peter, editor, Use andInterpretation of Laboratory Tests in Infectious Disease, 5^(th)edition, Specialty Laboratories, Santa Monica, Calif. 90404, 1998, pages1-271), since the compounds generally enhance Th1 immune responsesand/or reduce Th2 immune responses. Difficulty in treating someinfections, e.g., progressive toxoplasmic encephalitis, malaria,tuberculosis, leishmaniasis and schistosomiasis, often appear to beassociated with unwanted Th2 immune responses. Typically unwanted Th2immune responses are associated with, or caused by, increased expressionof one or more cytokines or interleukins such as IL-4 and IL-10.Administration of a formula 1 compound, or other compounds disclosedherein, will generally reduce the expression of one or more of theTh2-associated cytokines or interleukins. At the same time, thecompounds enhance the expression of one or more cytokines orinterleukins associated with Th1 immune responses. Because of theircapacity to modulate Th1 and Th2 immune responses, the compounds areuseful for a variety of clinical conditions, e.g., infection,immunosuppression or cancer, where an enhanced Th1 immune response isdesired. For example, in disseminated or diffuse tuberculosis, a reducedTh2 response would be desirable to allow a patient to slow progressionof the disease or to clear infected cells more efficiently.

An aspect of the invention provides embodiments where a formula 1compound and a glutathione reductase inhibitor such as buthathionesulfoximine [CH₃—(CH₂)₃—S(═O)(═NH)—(CH₂)₂—CHNH₂—C(O)—OH] areadministered to a subject to treat infections, e.g., a parasiteinfection such as malaria, Toxoplasma, Cryptosporidium, or to treat acancer or malignancy. The decreased supply of reduced glutathione mayenhance phagocytosis by macrophage, possibly due to enhanced oxidativedamage in infected cells or in replicating malignant cells.Alternatively, the use of a glutathione reductase inhibitor may resultin improved recognition of infected or malignant cells by the immunesystem. A formula 1 compound, such as BrEA, and buthathione sulfoximineare used, e.g., to enhance clearance of ring stage malaria from infectedcells or to enhance immune system recognition of malignant cellscompared to the use of the formula 1 compound alone. The infections andmalignancies where these embodiments apply are as described herein.

Another aspect of the invention provides for the use of a formula 1compound and a flavonoid, e.g., a naragin flavonoid, to enhance thebioavailability of the formula 1 compound. In these embodiments, the aneffective amount of a flavonoid is administered to a subject who isreceiving a formula 1 compound. Typically about 1-10 mg of flavonoid perkg of body weight is administered to the subject a flavonoid such asbavachinin A, didymin (isosakuranetin-7-rutinoside or neoponcirin),flavanomarein (isookanine-7-glucoside), flavanone azine, flavanonediacetylhydrazone, flavanone hydrazone, silybin, silychristin,isosilybin or silandrin. The flavonoid compound is typicallyadministered with the formula 1 compound or a few hours, e.g., about 1,2 or 3 hours, before the formula 1 compound is administered to thesubject.

Liposome formulations can be used to enhance delivery of the formula 1compound(s) to certain cell types such as tumor cells (see e.g., U.S.Pat. No. 5,714,163) or to cells of the reticuloendothelial system(“RES”). The RES includes macrophages, mononuclear phagocytic cells,cells lining the sinusoids of the spleen, lymph nodes, and bone marrow,and the fibroblastic reticular cells of hematopoietic tissues. Ingeneral, RES cells are phagocytic and they are targets for targeteddelivery of a formula 1 compound(s) in vitro or in vivo using liposomes,or other compositions or formulations. Thus, one can deliver formula 1compound to a neoplasm that is derived from reticuloendothelial tissue(reticuloendothelioma). The liposomes may also optionally comprise apeptide from an infectious agent such as a malaria parasite. Thepeptides may facilitate the generation of a MHC class II and B cellresponse.

Vaccine adjuvants. The compounds disclosed herein may also be used asvaccine adjuvants with immunogens or components of immunogeniccompositions to prepare antibodies capable of binding specifically tothe formula 1 compounds, their metabolic products which retainimmunologically recognized epitopes (sites of antibody binding) or tostandard antigens that are used for vaccination against, e.g.,infectious agents or malignant cells. The immunogenic compositionstherefore are useful as intermediates in the preparation of antibodiesthat bind to formula 1 compounds for use, e.g., in diagnostic, qualitycontrol, or the like, methods or in assays for the compounds or theirnovel metabolic products. In addition, the compounds are useful forraising antibodies against otherwise non-immunogenic polypeptides, inthat the compounds may serve as haptenic sites stimulating an immuneresponse.

The hydrolysis products of interest include products of the hydrolysisof the protected acidic and basic groups discussed above. In someembodiments the acidic or basic amides comprising immunogenicpolypeptides such as albumin, keyhole limpet hemocyamin and othersdescribed below generally are useful as immunogens. The metabolicproducts described above may retain a substantial degree ofimmunological cross reactivity with the compounds of the invention.Thus, the antibodies of this invention will be capable of binding to theunprotected compounds of the invention without binding to the protectedcompounds; alternatively the metabolic products, will be capable ofbinding to the protected compounds and/or the metabolic products withoutbinding to the protected compounds of the invention, or will be capableof binding specifically to any one or all three. The antibodiesdesirably will not substantially cross-react with naturally-occurringmaterials. Substantial cross-reactivity is reactivity under specificassay conditions for specific analytes sufficient to interfere with theassay results.

The immunogens of this invention contain the compound of this inventionpresenting the desired epitope in association with an immunogenicsubstance. Within the context of the invention such association meanscovalent bonding to form an immunogenic conjugate (when applicable) or amixture of non-covalently bonded materials, or a combination of theabove. Immunogenic substances include adjuvants such as Freund'sadjuvant, immunogenic proteins such as viral, bacterial, yeast, plantand animal polypeptides, in particular keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin or soybean trypsin inhibitor, andimmunogenic polysaccharides. Typically, the compound having thestructure of the desired epitope is covalently conjugated to animmunogenic polypeptide or polysaccharide by the use of a polyfunctional(ordinarily bifunctional) cross-linking agent. Methods for themanufacture of hapten immunogens are conventional per se. Any of themethods used heretofore for conjugating haptens to immunogenicpolypeptides or the like are suitably used here, taking into account thefunctional groups on the precursors or hydrolytic products which areavailable for cross-linking and the likelihood of producing antibodiesspecific to the epitope in question as opposed to the immunogenicsubstance.

Typically the polypeptide is conjugated to a site on the compound of theinvention distant from the epitope to be recognized.

The conjugates are prepared in conventional fashion. For example, thecross-linking agents N-hydroxysuccinimide, succinic anhydride or C₂₋₈alkyl-N═C═N—C₂₋₈ alkyl are useful in preparing the conjugates of thisinvention. The conjugates comprise a compound of the invention attachedby a bond or a linking group of 1-100, typically, 1-25, more typicallyabout 1-10 carbon atoms to the immunogenic substance. The conjugates areseparated from starting materials and by products using chromatographyor the like, and then are sterile filtered and vialed for storage.Synthetic methods to prepare hapten-carrier immunogens have beendescribed, see, e.g., G. T. Hermanson, Bioconjugate Techniques AcademicPress, 1996, pages 419-493.

The compounds of this invention are cross-linked for example through anyone or more of the following groups: a hydroxyl group, a carboxyl group,a carbon atom, or an amine group. Included within such compounds areamides of polypeptides where the polypeptide serves as anabove-described protecting group.

Animals are typically immunized against the immunogenic conjugates orderivatives and antisera or monoclonal antibodies prepared inconventional fashion.

In embodiments where the formula 1 compounds are used as adjuvants toenhance a subject's immune response to antigens such as proteins,peptides or virus or cell preparations, the formula 1 compound isadministered at about the same time that the antigen is delivered to thesubject, e.g., within about 7 days of when the antigen is administeredto the subject. In some embodiments, the formula 1 compound isadministered 1, 2, 3 or 4 days before the antigen is administered to thesubject. In other embodiments, the formula 1 compound is administered onthe same day that the antigen is administered to the subject. Inadditional embodiments, the formula 1 compound is administered 1, 2 or 3days after the antigen is administered. The formula 1 compound can beadministered to the subject using any of the formulations or deliverymethods described herein or in the references cited herein. Aspects ofthe invention include compositions or formulations that comprise aformula 1 compound, one or more excipients and an antigen or antigenpreparation such as disrupted cells or viruses or such as attenuatedviruses or a DNA vaccine.

Related embodiments include a method comprising administering to asubject (e.g., a mammal such as a human or a primate), or delivering tothe subject's tissues, an effective amount of a formula 1 compound and aspecific antigen. These methods are useful to enhance the subject'simmune response to the antigen. Immune responses that are enhancedinclude a mucosal immune response to an antigen such as a protein,peptide, polysaccharide, microorganism, tumor cell extract or lethallyradiated tumor or pathogen cells (e.g., antigens or cells from melanoma,renal cell carcinoma, breast cancer, prostate cancer, benign prostatichyperplasia, virus or bacteria, or other tumor or pathogen as disclosedherein). Aspects of these embodiments include enhancement of thesubject's immune response when an antigen or immunogen is administeredintranasally or orally. In these aspects, the formula 1 compound isadministered about simultaneously with the antigen or within about 3-72hours of antigen administration. The use of immune modulating agents toenhance immune responses to a vaccine has been described, e.g., U.S.Pat. No. 5,518,725.

Other uses for the formula 1 compound(s) include administering thecompound(s) to a subject who suffers from a pathological condition(s).The treatment may treat or ameliorate the source of the condition(s)and/or symptoms associated with the pathological condition(s) such asinfection with a pathogen(s) (viruses, bacteria, fungi), a malignancy,unwanted immune response, i.e., an immune response that causes pathologyand/or symptoms, e.g., autoimmune conditions or allergy or conditionssuch as hypoproliferation conditions, e.g., normal or impaired tissuegrowth, or wound healing or burn healing, or in immunosuppressionconditions, e.g., conditions characterized by an absence of a desiredresponse and/or an inadequate degree of a desired response.

Many cancers or malignancies are associated with an unwanted Th2 immuneresponse or a deficient Th1 response. An insufficient Th1 immuneresponse may play a role in the capacity of malignant cells to escapeimmune surveillance. These conditions include non-small cell lungcancer, bronchogenic carcinoma, renal cell cancer or carcinoma,lymphoma, glioma, melanoma, pancreatic or gastric adenocarcinoma, humanpapillomavirus associated cervical intraepithelial neoplasia, cervicalcarcinoma, hepatoma and cutaneous T-cell lymphoma (mycosis fungoides,Sezary syndrome).

In some of these embodiments, the subject's hyperproliferation ormalignant condition may be associated with one or more pathogens. Forexample hepatocellular carcinoma associated with HCV or HBV, Kaposi'ssarcoma associated with HIV-1 or HIV-2, T cell leukemia associated withHTLV I, Burkitt's lymphoma associated with Epstein-Barr virus orpapillomas or carcinoma associated with papilloma viruses (HPV 6, HPV11, HPV 16, HPV 18, HPV 31, HPV 45) or gastric adenocarcinoma or gastricMALT lymphoma associated with Helicobacter pylori infection. In otherembodiments, the formula 1 compound(s) is administered to a subject whohas a hyperproliferation condition that appears to not be associatedwith a pathogen, e.g., melanoma, or a cancer or precancer arising in thethroat, esophagus, stomach, intestine, colon, ovary, lung, breast orcentral nervous system.

In an exemplary embodiment, human patients suffering from melanoma ormelanoma precursor lesions are treated with a topical cream formulationcontaining 2-20% BrEA (w/w). The cream is applied to primary nevi(dysplastic nevi or common acquired nevi), primary cutaneous melanomas,secondary cutaneous melanomas and the skin surrounding the nevi ormelanomas. The areas to be treated are washed with soap or swabbed withan alcohol (e.g., ethanol or isopropanol) prior to administering thecream, when this is practical. About 0.1-0.4 g of cream, depending onthe size of the treated area, is applied once or twice per day pertreated region or lesion for about 10-20 days. The cream is leftundisturbed at the administration site for about 15-30 minutes beforethe patient resumes normal activity. Progression of the nevi andmelanomas is retarded in the majority of patients and significantregression is observed for some lesions. Following initial treatment,the formulation is administered every other day for at least 1 to 4months using the same dosing described for the initial round oftreatment. For these patients, standard therapy to treat precursorlesion or melanoma, e.g., dimethyl triazeno imidazole carboxamide ornitrosoureas (e.g., BCNU, CCNU), is optionally started or continuedaccording to the recommendations of the patient's doctor and with thepatient's informed approval. In cases where a tumor or precursor lesionis surgically removed and the site has sufficiently healed, the patientoptionally continues using the topical formulation at the site and theadjacent surrounding area every other day for at least 1 to 4 months. Insome of these embodiments, a formula 1 compound(s) is administered dailycontinuously as an oral composition or formulation, e.g., for a formula1 compound(s) that is a new compound per se. BrEA is optionally alsoadministered systemically using, e.g., a formulation described in theexamples below to deliver 1-5 mg/kg/day every other day for about 1 weekto about to 4 months, e.g., in the case of malignant melanoma.

Insufficient Th1 immune responses are often associated with viralinfection. Viral infections may arise from DNA or RNA viruses, e.g.,herpesviruses, hepadnaviruses, adenoviruses, retroviruses, togaviruses,aiphaviruses, arboviruses, flaviviruses, rhinoviruses, papillomavirusesand/or pestiviruses. Exemplary viruses have been described. See, forexample B. N. Fields, et al., editors, Fundamental Virology, 3^(rd)edition, 1996, Lippencott-Raven Publishers, see chapter 2 at pages23-57, including table 4 at pages 26-27, table 5 at pages 28-29, chapter17 at pages 523-539, chapters 26-27 at pages 763-916, chapter 32 atpages 1043-1108 and chapter 35 at pages 1199-1233. As used herein,retroviruses include human and animal viruses, e.g., HIV-1, HIV-2, LAV,human T-cell leukemia virus I (“HTLV I”), HTLV II, HTLV III, SIV, SHIV,FIV, FeLV. Additional viruses, including their genogroups, clades,isolates, strains and so forth, that may establish a virus infectioninclude human hepatitis C virus (“HCV”), human hepatitis B virus(“HBV”), human hepatitis A virus (“HAV”), duck hepatitis virus,woodchuck hepatitis virus, human (“HPV”, e.g., HPV 6, HPV 11, HPV 16,HPV 18, HPV 31, HPV 45) or animal papilloma viruses, Poliovirus, Herpessimplex virus 1 (“HSV-1”), Herpes simplex virus 2 (“HSV-2”), humanHerpesvirus 6 (“HHV-6”), human Herpesvirus 8 (“HHV-8”), Dengue virus(types 1-4), Western Equine Encephalitis Virus, Japanese EncephalitisVirus, Yellow Fever Virus and Bovine Viral Diarrhea Virus.

Other conditions where an immune imbalance or an excessive Th2 immuneresponse is involved include autoimmune diseases such as SLE (systemiclupus erythematosus), osteoporosis, multiple sclerosis, myastheniagravis, Graves disease, mite-associated ulcerative dermatitis,rheumatoid arthritis and osteoarthritis. Excessive Th2 immune responsesare also associated with an unwanted symptom or pathology, e.g.,fatigue, pain, fever or an increased incidence of infection, that isassociated with aging, allergy and inflammation conditions such asallergic bronchopulmonary aspergillosis in cystic fibrosis patients,atopic asthma, allergic respiratory disease, allergic rhinitis, atopicdermatitis, subepithelial fibrosis in airway hyperresponsiveness,chronic sinusitis, perennial allergic rhinitis, Crohn's disease(regional enteritis), ulcerative colitis, inflammatory bowel disease,fibrosing alveolitis (lung fibrosis).

Other clinical indications that have an association with or have asymptom(s) that is consistent with an excessive Th2 immune response,e.g., fatigue, pain, fever or an increased incidence of infection, areschizophrenia, acute myelitis, sarcoidosis, burns, trauma (e.g., bonefracture, hemorrhage, surgery) and immune responses toxenotransplantation. This common underlying immune component in at leastpart of the pathology of all of these conditions allows a single agentto be effectively used to treat the condition or to treat one or moresymptoms that are associated with insufficient Th1 responses or withexcessive Th2 responses. In all of the conditions where an insufficientTh1 response or an unwanted Th2 response is present, amelioration of oneor more symptoms associated with the condition is accomplished byadministering an effective amount of a formula 1 compound according tothe methods described herein. Thus, one may intermittently administer aformula 1 compound using a formulation and a route of administration asdescribed herein.

In some applications, the formula 1 compound(s) may directly and/orindirectly interfere with replication, development or cell-to-celltransmission of a pathogen such as a virus or a parasite (malaria).Improvement in a subject's clinical condition may arise from a directeffect on an infectious agent or on a malignant cell. Interference withcellular replication can arise from inhibition of one or more enzymesthat a parasite or an infected cell uses for normal replication ormetabolism, e.g., glucose-6-phosphate dehydrogenase, which affectscellular generation of NADPH (see, e.g., Raineri et al., Biochemistry1970 9: 2233-2243). This effect may contribute to cytostatic effectsthat some formula 1 compounds can have. Modulation of cellular enzymesexpression or activity may also interfere with replication ordevelopment of a pathogen, e.g., HIV or malaria parasites or withreplication or development of neoplastic cells, e.g., inhibition ofangiogenesis. Clinical improvement will also generally result from anenhanced Th1 immune response.

In other applications, embodiments are a method comprising contacting aformula 1 compound(s) with a cell(s), whereby the formula 1 compound(s)forms a complex with a steroid hormone receptor or results in themodulation of a biological activity. The steroid hormone receptor may bean orphan nuclear hormone receptor that displays a moderate or highbinding affinity for the formula 1 compound(s). In some embodiments, thesteroid receptor is a known steroid receptor. Biological effects frominteraction of a formula 1 compound and a receptor can lead tointerference with the replication or development of a pathogen or thecell(s) itself. For example, expression of HIV transcripts inHIV-infected cells may be altered. The receptor-formula 1 compoundcomplex may directly interfere with LTR-dependent transcription of HIVgenes, leading to reduced viral replication.

Invention embodiments include compositions comprising a partiallypurified or a purified complex comprising a formula 1 compound and asteroid receptor. Such a steroid receptor(s) may be an orphan steroidreceptor or a characterized steroid receptor, where either type bindsthe formula 1 compound with a moderate or high binding affinity, e.g.,less than about 0.5-10×10⁻⁶ M, usually less than about 1×10⁻⁷ M, or, forhigher affinity interactions, less than about 0.01-10×10⁻⁹ M. Theformula 1 compound(s) may also enhance immune responses such that bothimmune responses and altered intracellular conditions simultaneouslyexist to ameliorate one or more of the pathological conditions describedherein.

The formula 1 compounds may be used to identify receptors that modulatebiological responses, e.g., receptors that participate in effectingenhanced Th1 cytokine synthesis. Invention embodiments include a method,“Method 1”, which permits the determination of one or more effects of atest compound on a steroid receptor in various biological systems.Generally, the test compound is a formula 1 compound. Such systemsinclude cells containing a DNA construct that constitutively orinducibly expresses a steroid receptor(s) of interest, e.g., SXR, CARβ,RXR, PXR, PPARα or mixtures or dimers thereof, e.g., SXR/RXR. In otherbiological systems, the steroid receptor can be under thetranscriptional control of a regulatable promoter. Alternatively, theexpression another gene such as a steroid-inducible gene, e.g., asteroid-inducible cytochrome P-450. For this method, a source of steroidreceptors is generally combined with a means of monitoring them, e.g.,by measuring the transcription of a gene regulated by the receptor.Cells that comprise the steroid receptor and optional monitoring meansare sometimes referred to herein as the “biological system.” Sources ofsteroid receptors include cell lines and cell populations that normallyexpress the steroid receptor of interest and extracts obtained from suchcells. Another source for a useful biological system for purposes ofthis method is tissues from experimental animals that express thereceptor.

In one aspect, method 1 allows one to determine one or more effects of aformula 1 compound on a steroid receptor using a method that comprises(a) providing a biological system, e.g., a cell extract, cells ortissue, comprising cells having a plurality of steroid receptors thatcomprise monomers, homodimers or heterodimers that comprise a steroidreceptor, e.g., SXR, CAR-β, RXR, PPARα, PXR or dimers that comprise oneor more of these; (b) activating or inhibiting the plurality ofmonomers, homodimers or heterodimers that comprise the steroid receptorby contacting the cells with a steroid receptor (e.g., SXR, CARβ, RXR,PPARα or PXR) agonist or antagonist; (c) removing substantially all ofthe steroid receptor agonist or antagonist from the cells; (d)determining an activity of the plurality of monomers, homodimers orheterodimers that comprise the steroid receptor while in an activatedstate in the absence of agonist or antagonist; (e) exposing the cells tothe test compound; (f) determining at least one effect of the testcompound on the activity of the plurality of monomers, homodimers orheterodimers that comprise one or more of the steroid receptors whilethey remain substantially free of agonist or antagonist; and (g)optionally classifying the test compound as an agonist or an antagonistof the steroid receptor, or a neutral compound having little or nodetectable effect.

The effects that method 1 can measure include determining or measuringan effect on a gene whose expression is affected by the steroidreceptor. The gene could be a gene associated with a pathologicalcondition such as an infectious agent, an immune disorder or ahyperproliferation condition.

Thus, another aspect of method 1, “method 1A”, is determining if achemical not previously known to be a modulator of protein biosynthesiscan transcriptionally modulating the expression of a gene that encodes aprotein associated with the maintenance or treatment of one or moresymptoms of a pathological condition. This method comprises: (a)contacting a sample which comprises eucaryotic cells with a formula 1compound, wherein the eucaryotic cells comprise a plurality of steroidreceptor proteins and a DNA construct containing in 5′ to 3′ order (i) amodulatable transcriptional regulatory sequence of the gene encoding theprotein of interest, (ii) a promoter of the gene encoding the protein ofinterest, and (iii) a reporter gene which expresses a polypeptidecapable of producing a detectable signal, coupled to, and under thecontrol of, the promoter, under conditions such that the chemical, ifcapable of acting as a transcriptional modulator of the gene encodingthe protein of interest, causes a measurable or detectable signal to beproduced by the polypeptide expressed by the reporter gene; (b)quantitatively determining the amount of the signal so produced; and (c)optionally comparing the amount so determined with the amount ofproduced signal detected in the absence of any chemical being tested orupon contacting the sample with other chemicals so as to identify thechemical as one that causes a change in the detectable signal producedby the polypeptide, and determining whether the chemical specificallytranscriptionally modulates expression of the gene associated with themaintenance or treatment of one or more symptoms of the pathologicalcondition.

In conducting method 1A, one typically contacts a sample that contains apredefined number of identical or essentially identical eucaryotic cellswith a predetermined concentration of a compound of formula 1. Theeucaryotic cells comprise a DNA construct that is made usingconventional molecular biology methods and protocols. Generally the DNAconstruct contains in 5′ to 3′ order (i) a transcriptional regulatorysequence that participates in modulating expression of the gene that isassociated with maintaining or treating the pathological condition, (ii)the gene's promoter, and (iii) a reporter gene which expresses apolypeptide capable of producing a detectable signal, coupled to, andunder the control of, the promoter. The construct is maintained underconditions such that the formula 1 compound, if capable of acting as atranscriptional modulator of the gene encoding the protein of interest,causes a measurable or detectable signal to be produced by thepolypeptide expressed by the reporter gene. Once sufficient time forgeneration of a detectable response or signal has passed, one candetermine the amount of the signal produced. Typically the response orsignal is measured quantitatively, but a qualitative measurement can beuseful for rapid screening purposes.

For method 1A, one can also optionally compare the detectable signalwith the amount of produced signal that (i) one detects in the absenceof any formula 1 compound or (ii) when contacting the sample with otherchemicals, which identifies the formula 1 compound as a chemical thatcauses a change in the detectable signal the polypeptide produces. Onethen typically determines whether the formula 1 compound specificallytranscriptionally modulates expression of the gene associated with themaintenance or treatment of one or more symptoms of the pathologicalcondition.

Other aspects of the method 1 and 1A include a screening methodcomprising separately contacting each of a plurality of identical,essentially identical or different samples, each sample containing apredefined number of such cells with a with a predeterminedconcentration of each different formula 1 compound to be tested, e.g.,wherein the plurality of samples comprises more than about 1×10³ or morethan about 1×10⁴ samples or about 0.5-5×10⁵ samples. In other aspectsone determines the amount of RNA by quantitative polymerase chainreaction. In any of methods 1 or 1A, a formula 1 compound such as anyone of those described or named herein may be utilized.

Aspects of the invention include another method, “method 2”, whichcenters on identifying a gene whose expression is modulated by acandidate binding partner for infectious disease therapeutic agents.Typically the binding partner is a steroid receptor, e.g., a monomer,homodimer or heterodimer that comprises SXR, CAR-β, PXR, PPARα or RXR ora homolog or isoform thereof. The steroid receptor is typically presentas a complex that comprises, e.g., the formula 1 compound and theregulated gene's DNARS, which the steroid receptor, or a complex thatcomprises the steroid receptor, recognizes and specifically binds to.Such complexes can also comprise a transcription factor that binds tothe steroid receptor or to nucleic acid sequences adjacent to or nearthe DNARS. Exemplary transcription factors that may be present includeone or more of ARA54, ARA55, ARA70, SRC-1, NF-κB, NFAT, AP1, Ets, p300,CBP, p300/CBP, p300/CPB-associated factor, SWI/SNF and human homologs ofSWI/SNF, CBP, SF-1, RIP140, GRIP1 and Vpr. In general, one provides afirst and a second group of cells in vitro or in vivo and contacts thefirst group of cells with the infectious disease therapeutic agent, butdoes not contact the second group of cells in vitro or in vivo with theinfectious disease therapeutic agent. Recovering RNA from the cells, orgenerating cDNA derived from the RNA, is accomplished by conventionalprotocols. Analysis of the RNA, or cDNA derived from the RNA, from thefirst and the second group of cells identifies differences between them,which one can use to identify a gene whose regulation is modulated bythe candidate-binding partner for the infectious disease therapeuticagent or any DNARS associated with that gene.

An aspect of method 2 is determining the capacity of a formula 1compound to modulate, or participate in the modulation of, thetranscription of a gene associated with the maintenance or treatment ofone or more symptoms of a pathological condition. It is expected that ingeneral, the formula 1 compounds will cause an increase in thetranscription of such genes. The pathological condition is typically oneassociated with an infectious agent, e.g., virus, parasite or bacterium,but can also include an immune condition, e.g., an autoimmune conditionor an immune deficiency. The pathological condition may also be aninsufficient immune response to an infection or an insufficient responseto a hyperproliferation condition or malignancy. Other pathologicalconditions that one can apply the method to are inflammation conditions.

In some aspects, the formula 1 compounds used in method 2 will belabeled. Such compounds are prepared by conventional methods usingstandard labels, such as radiolabels, fluorescent labels or other labelsas described herein and in the cited references.

An embodiment of method 2 involves analyzing the RNA, or cDNA derivedfrom it, by subtraction hybridization. In this embodiment, the RNA orcDNA obtained from the first and second groups of cells is hybridizedand the resulting duplexes are removed. This allows recovery of nucleicacids that encode genes whose transcription is modulated by thecandidate-binding partner, which is usually a steroid receptor. One canuse conventional methods to amplify and obtain nucleic acid and proteinsequence information from the nucleic acids recovered by this method.The nucleic acid sequences that are transcriptionally induced orrepressed by the formula 1 compound are candidate-binding partners.

A transcriptionally induced gene(s) will be enriched in the group 1cells treated with the formula 1 compound, while any repressed gene(s)will be depleted or absent. In these embodiments, the RNA recoveredafter removal of duplexes is typically amplified by standard RT-PCR orPCR protocols. These protocols typically use specific sets of randomprimer pairs, followed by analysis of the amplified nucleic acids by gelelectrophoresis. Nucleic acids that are induced by the formula 1compound will appear as a band(s), usually duplex DNA, that is notpresent in the control or second set of cells. Nucleic acids that aretranscriptionally repressed by the formula 1 compound's binding partnerwill be depleted or absent in the first group of cells. Once such genecandidates are identified, they can be cloned and expressed and thecapacity of the DNARS associated with the gene to form a complex thatcomprises a candidate binding partner and an optionally labeled formula1 compound is analyzed by conventional methods, e.g., equilibriumdialysis, affinity chromatography using, e.g., the DNARS immobilized ona column, or coprecipitation of complexes that comprise an optionallylabeled DNARS and candidate binding partner using anti-binding partnerantibodies. Nucleic acid sequence analysis is usually used to identifyregions adjacent to the coding regions of the regulated gene to identifyany DNARS associated with the gene. The identity of a DNARS can beestablished by the binding to the DNARS of complexes that comprise acandidate binding partner, e.g., a steroid receptor, and optionally alsocomprise a formula 1 compound. The location and identity of the DNARScan be accomplished by DNA footprinting or other methods for detectingbinding interactions. The DNARS, the receptor or the formula 1 compoundcan be labeled in these variations of method 2.

In general, the second group of cells will be identical or essentiallyidentical to the first group of cells. In embodiments (for both methods1 and 2) where the cells are “essentially identical”, the first or thesecond group of cells may differ from each other by the presence orabsence of a DNA construct(s) that expresses (i) a steroid receptorand/or (ii) an easily detected protein, e.g., a β-galactosidase, aperoxidase, a phosphatase, or a chloramphenicol acetyltransferase, whosetranscriptional regulation is usually modulated by a steroid receptor.In these embodiments, the difference between the first and the secondgroup of cells is used to facilitate the analysis of the biologicaleffects of the formula 1 compound and the steroid receptor-bindingpartner. Groups of cells are considered “identical” if they do notdisplay known or obvious morphological or genetic differences.

Usually, the second group of cells will serve as a control, and theywill thus not be exposed to any formula 1 compound before obtaining theRNA or cDNA. But, for some embodiments, one can expose the second groupof cells to a known agonist or antagonist of the steroidreceptor-binding partner. This allows one to compare the potency of theformula 1 compound with the potency of the agonist or antagonist.

In other embodiments, one can modify method 2 by providing a third groupof cells, which is optionally used as an untreated control when thesecond group of cells is treated with a steroid receptor agonist orantagonist. In these embodiments, one will typically compare the effectof the formula 1 compound and the agonist or the antagonist of theexpression of a gene or DNA construct. The DNA construct would comprisea promoter or other regulatory sequences that are subject totranscriptional modulation, usually increase transcription, by theformula 1 compound in concert with its binding partner.

Exemplary mammalian and other steroid receptors, including orphansteroid receptors, their homologs, isoforms and co-factors (e.g.,co-repressors, transcription factors, gene promoter regions orsequences) that these complexes can comprise are steroidogenic factor-1(SF-1), chicken ovalbumin upstream promoter-transcription factor(COUP-TFI) and its mammalian homologs, silencing mediator for retinoidand thyroid hormone receptor (SMRT) and its mammalian homologs, NF-E3,COUP-TFII and its mammalian homologs, testicular orphan receptor TR2,thyroid hormone α1 (TR α1), retinoid X receptor α, TR α1/RXR αheterodimer, direct repeat-4 thyroid hormone response element (DR4-TRE),estrogen receptor (ER), estrogen receptor related α (ERRα), estrogenreceptor related β (ERRβ), steroid xenobiotic receptor (SXR), hepatocytenuclear factor 4 (HNF-4), hepatocyte nuclear factor 3 (HNF-3), liver Xreceptors (LXRs), LXRα, estrogen receptor α (ERα), constitutiveandrostane receptor-β (CAR-β), RXR/CAR-β heterodimer, short heterodimerpartner (SHP), SHP/ERα heterodimer, estrogen receptor β, SHP/ERβheterodimer, testicular orphan receptor TR4, TR2/TR4 heterodimer,pregnane X receptor (PXR) and isoforms, cytochrome P-450 monooxygenase3A4 gene promoter region and isoforms, HNF-4/cytochrome P-450monooxygenase 3A4 gene promoter region and isoforms complex, HIV-1 longterminal repeat (LTR), HIV-2 LTR, TR2/HIV-1 LTR complex, TR4/HIV-1 LTRcomplex, TR4/HIV-1 LTR complex, TR α1/TR4/HIV-1 LTR complex, TR2isoforms (TR2-5, TR7, TR9, TR11), DAX-1, DAX-1/steroidogenic acuteregulatory protein gene promoter region, RevErb, Rev-erbA α, Rev-erb β,steroid receptor coactivator amplified in breast cancer (AIB 1),p300/CREB binding protein-interacting protein (p/CIP), thyroid hormonereceptor (TR, T3R), thyroid hormone response elements (T3REs),constitutive androstane receptor (CAR), Xenopus xSRC-3 and mammalian(human) homologs, TAK1, TAK1/peroxisome proliferator-activated receptorα (PPARα) complex, PPARα/RXRα complex, TAK-1/RIP-140 complex, retinoicacid receptor (RAR), RARβ, TR4/RXRE complex, SF-1/steroid hydroxylasegene promoter region, SF-1/oxytocin gene promoter region, SF-1/ACTHreceptor gene promoter region, rat Ear-2 and mammalian homologs, humanTR3 orphan receptor (TR3), RLD-1, OR-1, androgen receptor,glucocorticoid receptor, estrogen receptor, progesterone receptor,mineralcorticoid receptor, OR1, OR1/RXRα complex, TIF-1, CBP/P300complex, TRIP1/SUG-1 complex, RIP-140, SRC1α/P160 complex andTIF-2/GRIP-1 complex, RAR/N-CoR/RIP13 complex, RAR/SMRT/TRAC-2 complex,and the DNARS 5′ AGGTCANAGGTCA 3′ or 5′ TGCACGTCA 3′. One of thesecomplexes can be included in invention methods when, e.g., they areperformed in cell-free assays. Formation of these complexes in cells isfacilitated by inserting into the cells a DNA construct(s) thatexpresses one or more of these proteins, e.g., mammalian or yeast cellscontaining a stable DNA construct or a construct used for transienttransfection assays. Methods to perform assays or to induce biologicalresponses in vitro or in vivo using the formula 1 compounds as agonists,antagonists or as reference standards are essentially as described, see,e.g., U.S. Pat. Nos. 5,080,139, 5,696,133, 5,932,431, 5,932,555,5,935,968, 5,945,279, 5,945,404, 5,945,410, 5,945,412, 5,945,448,5,952,319, 5,952,371, 5,955,632, 5,958,710, 5,958,892, 5,962,443;International Publication Numbers WO 96/19458, WO 99/41257, WO 99/45930.The complexes or assay systems, that comprise a formula 1 compound andthat are employed in the practice of these methods are included asaspects of the invention.

The formula 1 compounds typically interact with one or more biologicalligands to effect a biological response. To facilitate theidentification of candidate binding partners for the formula 1compounds, one can use a radiolabeled formula 1 compound that is linkedto a support, usually a solid support, as a means to recover thecandidate binding partners. The formula 1 compound can be linked to thesupport through, e.g., the 3-, 7-, 16- or 17-position of the steroidnucleus. Linking agents are known for such uses and includehomobifunctional and heterobifunctional agents, many of which arecommercially available. The linker one uses will typically compriseabout 2-20 linked atoms. The linked atoms usually comprise mostlycarbon, with one, two or three oxygen, sulfur or nitrogen atoms thatreplace one or more carbon atoms. One can use a cDNA expression librarythat one has made from suitable cells or tissues as a source ofcandidate binding partners. The cells or tissues can be obtained from amammalian or a vertebrate host, e.g., human, mouse, bird, primate, orfrom other sources, e.g., insects (e.g., Drosophila), otherinvertebrates (e.g., yeast, bacteria, Mycoplasma sp., Plasmodium sp.,Tetrahymena sp., C. elegans) or other organism groups or species listedherein or in the cited references. Suitable tissues include skin, livertissue or cells, including hepatocytes and Kupfer cells, fibrocytes,monocytes, dendritic cells, kidney cells and tissues, brain or othercentral nervous system cells or tissues, including neurons, astrocytesand glial cells, peripheral nervous system tissues, lung, intestine,placenta, breast, ovary, testes, muscle, including heart or myocytetissue or cells, white blood cells, including T cells, B cells, bonemarrow cells and tissues, lymph tissues or fluids and chondrocytes.

Typically a candidate-binding partner that one isolates from a non-humansource will have a human homolog that has similar binding properties forthe formula 1 compound. Non-human candidate-binding partners can thus beused to facilitate recovery of the human homologs, e.g., by preparingantiserum for precipitating the human homolog from a solution thatcomprises the human homolog or by comparing the sequence of thenon-human candidate-binding partner with known human gene sequences.Once a source of the candidate-binding partner is obtained, it can becontacted with labeled formula 1 compound, usually radiolabeled with,e.g., ¹⁴C or ³H, and complexes that comprise the labeled formula 1compound and the candidate-binding partner is recovered using, e.g.,affinity chromatography or antibody precipitation methods. The recoveryof the complex provides a source of at least partially purifiedcandidate-binding partner, i.e., the candidate-binding partner isenriched, e.g., at least 10-fold enriched, or at least 100-foldenriched, or at least 500-fold enriched, compared to its abundance inthe original candidate-binding partner source material.

Aspects of the invention include a composition comprising a partiallypurified (purified at least about 2-fold to about 10-fold relative tonatural sources, e.g., cells or a cell lysate) comples or a purified(purified at least about 20-fold to about 5000-fold relative to naturalsources, e.g., cells or a cell lysate) complex (where the partiallypurified or purified complex is optionally isolated) comprising aformula 1 compound and a steroid receptor, a serum steroid-bindingprotein (e.g., human serum albumin, α1-acid glycoprotein, sexhormone-binding globulin, testosterone-binding globulin,corticosteroid-binding globulin, androgen binding protein (rat)) oranother binding partner, e.g., transcription factor or DNARS. An aspectof these compositions includes a product produced by the process ofcontacting the partially purified or the purified composition with oneor more cells, one or more tissues, plasma or blood.

Other aspects include a method to determine a biological activity of aformula 1 compound comprising: (a) contacting the formula 1 compound(s)with a cell or cell population; (b) measuring one or more of (i) acomplex between a binding partner and the formula 1 compound, (ii)proliferation of the cell or cell population, (iii) differentiation ofthe cell or cell population (iv) an activity of a protein kinase C, (v)a level of phosphorylation of a protein kinase C substrate, (vi)transcription of one or more target genes, (vii) inhibition of thecellular response to steroids, e.g., glucocorticoids, (viii) inhibitionof steroid-induced transcription, e.g., glucocorticoids, sex steroids or(ix) inhibition of HIV LTR-driven transcription; and (c) optionallycomparing the result obtained in step (b) with an appropriate control.Aspects of this embodiment include (i) the method wherein the bindingpartner is a steroid receptor, a transcription factor or a DNARS, (ii)the method wherein the biological activity determined is a modulatingactivity of the formula 1 compound for replication or cytopathic effectsassociated with a retrovirus, a hepatitis virus or a protozoan parasite,(iii) the method wherein the biological activity determined is amodulating activity of the formula 1 compound for replication,cytopathic effects associated with the retrovirus, the hepatitis virusor the protozoan parasite or the biological activity determined ismetabolism (assay by ³H-thymidine uptake or other assay as referenced ordescribed herein) of a cell or cell population comprising NK cells,phagocytes, monocytes, macrophages, basophils, eosinophils, dendriticcells, synoviocytes, microglial cells, fibrocytes, transformed(neoplastic) cells, virus-infected cells, bacteria-infected cells orparasite-infected cells, and (iv) the method wherein the target gene isa virus gene, a bacterial gene, a parasite gene, a gene associated withcancer, e.g., wherein the virus gene is a DNA or an RNA polymerase gene,a reverse transcriptase gene, an envelope gene, a protease gene or agene associated with viral nucleic acid replication or a viralstructural gene.

An embodiment is a method comprising contacting a complex that comprisesa steroid receptor and a formula 1 compound with a transactivatorprotein, whereby a complex comprising the steroid receptor protein, theformula 1 compound and the transactivator protein forms, wherein thetransactivator protein is in (1) a cell or tissue extract (e.g., nuclei,lysate containing nuclei or lysate without nuclei from a cell(s) ortissue(s)), (2) a partially purified or purified cell or tissue extract,(3) a cell(s) in tissue culture or (4) a cell(s) in a subject, where anyof (1)-(4) optionally comprises a target gene (native gene or introducedby standard gene manipulation techniques) whose level of expression isoptionally assayed after the complex forms. In some of theseembodiments, the transactivator protein is partially purified orpurified and is in the cell or tissue extract or the partially purifiedor purified cell or tissue extract. The transactivator protein may beTIF-1, CBP/P300, TRIP1/SUG-1, RIP-140, SRQ1α/P160, or TIF-2/GRIP-1. Inany of these embodiments the complex comprising the steroid receptorprotein, the formula 1 compound and the transactivator protein mayincrease or decrease transcription of the target gene compared to asuitable control (e.g., control under same conditions, but lacking anyadded compound that corresponds to the formula 1 compound, or whereanother compound (e.g., a steroid that is known to bind to the steroidreceptor) is used as a benchmark or reference standard against whichaltered target gene expression is measured). In these methods, thetarget gene may be a pathogen gene (e.g., virus, bacterium, parasite,fungus, yeast) or a gene associated with a pathological condition(autoimmunity, inflammation, hyperproliferation).

The formula 1 compounds are suitable for use in certain describedmethods that use steroids to modulate biological activities in cells ortissues. For example, a formula 1 compound(s) can be used to selectivelyinteract with specific steroid receptors or steroid orphan receptor, ortheir subtypes, that are associated with a pathological condition(s) ina subject, essentially as described in U.S. Pat. No. 5,668,175. In theseapplications, the formula 1 compound may act as a ligand for thereceptor to modulate abnormal expression of a gene product(s) thatcorrelates with the pathological condition (a steroid hormone responsivedisease state). Such genes are normally regulated by steroid hormones.In other applications, one can use the formula 1 compounds to screen forligands that bind to a steroid receptor or steroid orphan receptor andone or more transcription factors (or cofactors) such as AP-1 and/orwith a DNA sequence(s), essentially as described in U.S. Pat. No.5,643,720. Similarly, the formula 1 compounds can be used essentially asdescribed in U.S. Pat. Nos. 5,597,693, 5,639,598, 5,780,220, 5,863,733and 5,869,337. In some of these embodiments, the formula 1 compound(s)is labeled to facilitate its use. Suitable labels are known in the artand include radiolabels (e.g., ³H, ¹⁴C, ³²P, ³⁵S, ¹³¹I, ⁹⁹Tc and otherhalogen isotopes), fluorescent moieties (e.g., fluorescein, resorufin,Texas Red, rhodamine, BODIPY, arylsulfonate cyanines), chemiluminescentmoieties (e.g., acridinium esters), metal chelators, biotin, avadin,peptide tags (e.g., histidine hexamer, a peptide recognized bymonoclonal or polyclonal antibodies), covalent crosslinking moieties.One prepares the labeled compounds according to known methods.

Methods suitable to measure the biological effects of various compounds,e.g., activation, on immune system cells (e.g., NK cells, phagocytes,monocytes, macrophage, neutrophils, eosinophils, dendritic cells,synoviocytes, microglial cells, fibrocytes) have been described, e.g.,Jakob et al., J. Immunol. 1998 161:3042-3049, Pierson et al., Blood 199687:180-189, Cash et al., Clin. Exp. Immunol. 1994 98:313-318, Monick etal., J. Immunol. 1999 162:3005-3012, Rosen et al., Infect Immun. 199967:1180-1186, Grunfeld et al., J. Lipid Res. 1999 40:245-252, Singh etal., Immunol. Cell Biol. 1998 76:513-519, Chesney et al., Proc. Natl.Acad. Sci. USA 1997 94:6307-6312, Verhasselt et al., J. Immunol. 1999162:2569-2574, Avice et al., J. Immunol. 1999 162:2748-2753, Cella etal., J. Exp. Med. 1999 189:821-829, Rutalt et al., Free Radical Biol.Med. 1999 26:232-238, Akbari et al., J. Exp. Med. 1999 189:169-178,Hryhorenko et al., Immunopharmacology 1998 40:231-240, Fernvik et al.,Inflamm. Res. 1999 48:28-35, Cooper et al., J. Infect. Dis. 1999179:738-742, Betsuyaku et al., J. Clin. Invest. 1999 103:825-832, Brownet al., Toxicol. Sci. 1998 46:308-316, Sibelius et al., Infect. Immunol.1999 67:1125-1130. The use of formula 1 compounds in such methods areaspects of the invention and they permit, e.g., measurement of thebiological effects of formula 1 compounds on genes whose expression isregulated by the formula 1 compound and the steroid receptor.

Embodiments include any of the methods described above, e.g., method 1,wherein the cells or biological system comprises NK cells, phagocytes,monocytes, macrophage, neutrophils, eosinophils, dendritic cells,synoviocytes, microglial cells, glial cells, fibrocytes or hepatocytes,that optionally comprise a DNA construct that expresses one or twocloned steroid receptors. The method optionally analyzes the effect of aformula 1 compound on the cells compared to controls. Controls includethe use of a known agonist or antagonist for the steroid receptor or thecomparison of cells exposed to a formula 1 compound with control cells(usually the same cell type as the treated cells) that are not exposedto the formula 1 compound. A response, e.g., activation of the steroidreceptor can be measured by known assays compared to controls.

The formula 1 compound will, in some cases modulate (increase ordecrease) transcription of one or more genes in the cells. In othercases, the formula 1 compound will enhance lysosome movement in one ormore of the subject's NK cells, phagocytes, monocytes, macrophages,neutrophils, eosinophils, dendritic cells synoviocytes, microglial cellsor fibrocytes. Such effects will typically be mediated directly orindirectly through steroid receptors that act to modulate genetranscription, e.g., cause enhances protein kinase C (PKC) activity inthe cells used in the assay, e.g., PKCα, PKCβ, PKCγ or PKCζ.

Other related embodiments are a composition comprising a partiallypurified or a purified complex comprising a formula 1 compound and asteroid receptor, a serum steroid-binding protein (e.g., human serumalbumin, α1-acid glycoprotein, sex hormone-binding globulin,testosterone-binding globulin, corticosteroid-binding globulin, androgenbinding protein (rat)) or another binding partner, e.g., transcriptionfactor or DNARS. An aspect of these compositions includes a productproduced by the process of contacting the partially purified or thepurified composition with one or more cells, one or more tissues, plasmaor blood.

Another embodiment comprises a method to determine a biological activityof a formula 1 compound comprising: (a) contacting the formula 1compound(s) with a cell or cell population; (b) measuring one or more of(i) a complex between a binding partner and the formula 1 compound, (ii)proliferation of the cell or cell population, (iii) differentiation ofthe cell or cell population (iv) an activity of a protein kinase C, (v)a level of phosphorylation of a protein kinase C substrate, (vi)transcription of one or more target genes, (vii) inhibition of thecellular response to steroids, e.g., glucocorticoids, (viii) inhibitionof steroid-induced transcription, e.g., glucocorticoids, sex steroids or(ix) inhibition of HIV LTR-driven transcription; and (c) optionallycomparing the result obtained in step (b) with an appropriate control.Aspects of this embodiment include (i) the method wherein the bindingpartner is a steroid receptor, a transcription factor or a DNARS, (ii)the method wherein the biological activity determined is a modulatingactivity of the formula 1 compound for replication or cytopathic effectsassociated with a retrovirus, a hepatitis virus or a protozoan parasite,(iii) the method wherein the biological activity determined is amodulating activity of the formula 1 compound for replication,cytopathic effects associated with the retrovirus, the hepatitis virusor the protozoan parasite or the biological activity determined ismetabolism (assay by ³H-thymidine uptake or other assay as referenced ordescribed herein) of a cell or cell population comprising NK cells,phagocytes, monocytes, macrophages, basophils, eosinophils, dendriticcells, synoviocytes, microglial cells, fibrocytes, transformed(neoplastic) cells, virus-infected cells, bacteria-infected cells orparasite-infected cells, and (iv) the method wherein the target gene isa virus gene, a bacterial gene, a parasite gene, a gene associated withcancer, e.g., wherein the virus gene is a DNA or an RNA polymerase gene,a reverse transcriptase gene, an envelope gene, a protease gene or agene associated with viral nucleic acid replication or a viralstructural gene.

Another embodiment is a method comprising contacting a complex thatcomprises a steroid receptor and a formula 1 compound with atransactivator protein, whereby a complex comprising the steroidreceptor protein, the formula 1 compound and the transactivator proteinforms, wherein the transactivator protein is in (1) a cell or tissueextract (e.g., nuclei, lysate containing nuclei or lysate without nucleifrom a cell(s) or tissue(s)), (2) a partially purified or purified cellor tissue extract, (3) a cell(s) in tissue culture or (4) a cell(s) in asubject, where any of (1)-(4) optionally comprises a target gene (nativegene or introduced by standard gene manipulation techniques) whose levelof expression is optionally assayed after the complex forms. In some ofthese embodiments, the transactivator protein is partially purified orpurified and is in the cell or tissue extract or the partially purifiedor purified cell or tissue extract. The transactivator protein may beTIF-1, CBP/P300, TRIP1/SUG-1, RIP-140, SRC1α/P160, or TIF-2/GRIP-1. Inany of these embodiments the complex comprising the steroid receptorprotein, the formula 1 compound and the transactivator protein mayincrease or decrease transcription of the target gene compared to asuitable control (e.g., control under same conditions, but lacking anyadded compound that corresponds to the formula 1 compound, or whereanother compound (e.g., a steroid that is known to bind to the steroidreceptor) is used as a benchmark against which altered target geneexpression is measured). In these methods, the target gene may be apathogen gene (e.g., virus, bacterium, parasite, fungus, yeast) or agene associated with a pathological condition (autoimmunity,inflammation, hyperproliferation).

The biological effects observed while performing the methods describedherein are expected to usually involve the formation of complexes thatcontain two or more components. These components can include one or moretranscription factors or co-regulators or co-repressors of transcriptionand their homologs and isoforms. These factors and complexes containingthem include members of the steroid receptor coactivator-1 family(SRC-1, SRC-1/serum response factor), NF-κB, NFAT, p300, CBP, p300/CBP,p300/CPB-associated factor, SWI/SNF and human and other homologs, BRG-1,OCT-1/OAF, AP1, Ets, androgen receptor associated protein 54 (ARA54),androgen receptor associated protein 55 (ARA55), androgen receptorassociated protein 70 (ARA70), RAC3/ACTR, CREB-binding protein (CPB),SRC-1α, receptor interacting protein-140 (RIP-140), transcription factoractivator protein-1, activation function-2, glucocorticoidreceptor-interacting protein-1 (GRIP-1), receptor interactingprotein-160 (RIP-160), suppressor of gal4D lesions (SUG-1),transcription intermediary factor-1 (TIF-1), transcription intermediaryfactor-2 (TIF-2), SMRT, N-CoR, N-CoA-1, p/CIP, stroidogenic factor-1(SF-1), p65 (ReIA), and Vpr encoded by the human immunodeficiency virusand its isoforms and homologs. One or more of these factors can bepresent in complexes that comprise a formula 1 compound and a steroidreceptor, such as SXR, PPARα, CAR-β, RXR and/or PXR.

In a related embodiment, a formula 1 compound is used to exert acytostatic effect on mammalian cells in vitro. Typically such cells arelymphoid cells, e.g., T cell populations from, e.g., blood or organsthat are rich in lymphoid cells (e.g., spleen, lymph tissue or nodes),or transformed T cell lines. Such activity provides an estimate of thepotency of formula 1 compounds to mediate immunological effects, such asenhancing Th1 immune responses or suppressing expression of one or moreTh2-associated cytokines. Thus, an invention method comprises (a)contacting a formula 1 compound and lymphoid cells in vitro, (b)determining the degree of cytostasis that the compound exerts toidentify a cytostatic compound and (c) optionally administering thecytostatic compound to an immune suppressed subject to determine theeffect of the compound on one or more of the subject's immune responsesas described herein, e.g., enhanced Th1 cytokine or cell response ordecreased Th2-associated cytokine expression. Typically, such methodsare conducted using a range of formula 1 compound concentrations andsuitable controls, such as a known cytostatic agent or a blank thatcontains solvent that lacks the formula 1 compound. Inhibition of cellproliferation is measured by standard methods. Methods to measure thecytoststic effects of the compounds includes measuring viable cellnumbers in treated and untreated cultures or by measuring DNA synthesisusing e.g., ³H-thymidine incorporation into DNA in treated and untreatedcultures. Typical ranges of formula 1 concentrations in the cell growthmedium are about 0.1 μM to about 100 μM, using about 4-6 differentconcentrations of compounds with a fixed number of cells (e.g., about0.4×10⁵ to about 5×10⁵). The formula 1 compound is left in contact withthe cells in tissue culture for a sufficient time to observe cytostasis,e.g., about 16 hours to about 6 days, typically about 24-72 hours. Inthese embodiments, one may optionally screen for modulation of abiological activity of a steroid receptor, e.g., activation of PPARα,which may be associated with the cytostasis the compound induced.

In some applications, the formula 1 compound(s) appears to bring aboutan improvement of one or more of the symptoms associated with aninfection or a condition. For example, treatment of subjects who areimmune suppressed, e.g., from a retrovirus infection, cancerchemotherapy or other cause, generally show improvement of one or moreassociated symptoms, such as weight loss, fever, anemia, fatigue orreduced infection symptoms that are associated with a secondaryinfection(s), e.g., HSV-1, HSV-2, papilloma, human cytomegalovirus(“CMV”), Pneumocystis (e.g., P. carinii) or Candida (C. albicans, C.krusei, C. tropicalis) infections. The formula 1 compounds are alsouseful to facilitate immune system recovery in autologous bone marrowtransplant or stem cell transplant situations. In some embodiments, theformula 1 compound(s) is administered as a nonaqueous liquid formulationas described herein or the formula 1 compound(s) is administeredaccording to any of the intermittent dosing protocols described hereinusing a solid or liquid formulation(s). In the case of a subject who hasa retroviral infection with symptoms that include one or more of, arelatively low CD4 count (e.g., about 10-200, usually about 20-100), oneor more additional pathogen infections (HSV-1, HSV-2, HHV-6, HHV-8, CMV,HCV, a HPV, P. carini or Candida infection) and one or more of anemia,fatigue, Kaposi's sarcoma, fever or involuntary weight loss (at leastabout 5% of body weight), administration of about 0.1 to about 10mg/kg/day (usually about 0.4 to about 5 mg/kg/day) of a formula 1compound(s) to the subject typically results in noticeable improvementof one or more of the symptoms within about 1-4 weeks. In otherembodiments, the formula 1 compound(s) is administered to a subject whohas a condition that appears to be associated with a viral infection,e.g., pneumonia or retinitis associated with CMV, nasopharyngealcarcinoma or oral hairy leukoplakia associated with Epstein-Barr virus,progressive pancephalitis or diabetes associated with Rubella virus oraplastic crisis in hemolytic anemia associated with Parvovirus 19.

In an exemplary embodiment, human patients infected with HCV are dosedwith an aqueous isotonic α-cyclodextrin or β-cyclodextrin formulationcontaining about 20 mg/mL BrEA. The formulation is deliveredintravenously in a single daily dose or two subdoses per day. Thepatients are dosed with 1 to 10 mg/kg/day for 4 to 10 days, followed byno dosing for 5 to 30 days, followed by dosing again with thecyclodextrin formulation for 4 to 10 days. The dosing regimen isrepeated one, two or more times. Clinical markers for HCV infection arefollowed during treatment, e.g., viral nucleic acid in the blood orplasma, liver enzyme levels in the blood or plasma (e.g., AST/SGOT,ALT/SGPT, alkaline phosphatase). For these patients, a standard anti-HCVtreatment(s), e.g., interferon and/or ribavirin, is optionally startedor continued according to the recommendations of the patient's doctorand with the patient's informed approval. In some of these embodiments,a formula 1 compound(s) is administered daily continuously as acomponent in an oral or parenteral composition or formulation, e.g., fora formula 1 compound(s) that is a new compound per se. BrEA isoptionally also administered systemically using, e.g., the formulationof example 1 to deliver 1-5 mg/kg/day every other day for about 1 to 4months, or an oral formulation to deliver about 5-40 mg/kg/day everyother day for about 1 to 4 months.

In any of the embodiments disclosed herein, one can optionallyadminister an additional therapeutic treatment in conjunction with,i.e., before, during or after, administration of a formula 1 compound(s)to a subject(s). For example, in subjects who have a viral or parasiteinfection and are in the course of administration of a formula 1compound, other treatments can also be administered to the subject,e.g., nucleoside analogs for viral infections or chloroquine formalaria. Such additional treatments will typically include standardtherapies for the subject's pathological condition(s), but they can alsoinclude experimental or other treatments. For example, one cancoadminister vitamins (multivitamins, individual vitamins), antioxidantsor other agents (vitamin E, allopurinol), nutritional supplements(liquid protein or carbohydrate preparations) or other therapies as thepatient's medical condition warrants and the patient's doctorrecommends. Any of these additional treatments can be coupled with theadministration of any of the formula 1 compounds, e.g., BrEA, an ester,carbamate, carbonate or amino acid or peptide conjugate thereof, in anyof the embodiments described herein.

Such additional treatments are apparent to the skilled artisan. Suchtreatments are selected based on the condition(s) to be treated,cross-reactivities of ingredients and pharmaco-properties of thecombination. For example, when treating retroviral infections in a humanor other subject, the formula I compounds are combined with one or morereverse transcriptase inhibitors, protease inhibitors, antibiotics oranalgesics. Suitable formula 1 compounds include those described, e.g.,in compound groups 1 through 42-25-20-6 and elsewhere herein. Exemplaryreverse transcriptase inhibitors are AZT, 3TC, D4T, ddI, ddC, adefovirdipivoxil,9-[2-(R)-[[bis[[(isopropoxycarbonyl)oxy]methoxy]-phosphinoyl]methoxy]propyl]adenine,(R)-9-[2-(phosphonomethoxy)propyl]-adenine and adefovir. Exemplaryprotease inhibitors are indinavir, nelfinavir, ritonavir, crixivan andsequanavir. Fusion inhibitors may also be used, e.g., HIV fusioninhibitors. When treating viral infections of the respiratory system orother systems, e.g., hepatitis C virus (“HCV”) or influenza virusinfection (e.g., influenza A or B), the compositions of the inventionare optionally used in conjunction with antivirals (such asγ-interferon, amantidine, rimantadine, ribavirin or compounds disclosedin U.S. Pat. No. 5,763,483 (especially compounds recited in claims 1 and2) and U.S. Pat. No. 5,866,601), mucolytics, expectorants,bronchodilators, antibiotics, antipyretics, or analgesics.

In some embodiments, formula 1 compound(s) are administered to subjectswho have a parasite or bacterial infection, to slow the progression ofinfection, interfere with replication or development of the infectiousagent or to ameliorate one or more of the associated symptoms, e.g.,weight loss, anemia or secondary infections. Parasites are malariaparasites, sleeping sickness parasites and parasites associated withgastrointestinal infections. Parasites and bacteria include species,groups, genotypes, strains or isolates of gastroentestinal helminths,microsporidia, isospora, cryptosporidia (Cryptosporidium parvum),Mycobacterium (M. avium, M. bovis, M. leprae, M. tuberculosis, M.pneumoniae. M. penetrans), Mycoplasma (M. fermentans, M. penetrans, M.pneumoniae), Trypanosoma (T. brucei, T. gambiense, T. cruzi, T. evansi),Leishmania (L. donovani, L. major, L. braziliensis), Plasmodium (P.falciparum, P. knowlesi, P. vivax, P. berghei), Ehrlichia (E. canis, E.chaffeensis, E. phagocytophila, E. equi, E. sennetsu), Babesia microti,Haemophilus (H. somnus, H. influenzae), Brucella (B. militensis, B.abortus), Bartonella (B. henselae), Bordetella (B. bronchiseptica, B.pertussis), Escherichia (E. coli), Salmonella (S. typhimurium), Shigella(S. flexneri), Pseudomonas (P. aeruginosa), Neisseria (N. gonorrhoeae,N. meningitidis), Streptococcus, Staphylococcus (S. aureus), Rickettsia(R. rickettsii), Yersinia (Y. enterocolitica), Legionella pneumonia andListeria (L. monocytogenes).

One or more of the invention intermittent dosing protocols or one ormore of the liquid non-aqueous formulations described herein can beapplied by routine experimentation to any of the uses or applicationsdescribed herein. For a formula 1 compound(s) that is a new compound perse, the compound(s) can be administered to a subject according to aninvention intermittent dosing protocol(s) or by other protocols, e.g.,continuous daily dosing of a single dose or two or more subdoses perday. In addition any of the formula 1 compounds, e.g., one or moreformula 1 compounds that are new per se, can be present in any solid orliquid formulation described herein. These formulations and dosingprotocols can be applied by routine experimentation to any of the usesor applications described herein.

Numbered embodiments. Several aspects of the invention and relatedsubject matter includes the following numbered embodiments.

In some aspects, the invention relates to non-aqueous liquidformulations that comprise a formula 1 compound. Exemplary embodimentsare as follows.

1. A composition comprising one or more compounds of formula 1 orformula 2 and one or more nonaqueous liquid excipients, wherein thecomposition comprises less than about 3% v/v water.

2. The composition of embodiment 1 wherein the one or more formula 1compounds has the structure

wherein R⁷ and R⁹ independently are —CHR¹⁰—, —CH₂—, —CH═, —O—, —S— or—NH—, wherein R¹⁰ iS —OH, —SH, C₁₋₁₀ optionally substituted alkyl, C₁₋₁₀optionally substituted alkoxy, C₁₋₁₀ optionally substituted alkenyl orC₁₋₁₀ optionally substituted alkynyl; and R⁸ is —CH₂—, —O—, —S— or —NH—,wherein hydrogen atoms at the 5 (if present), 8, 9 and 14 positionsrespectively are α.α.α.α (i.e., 5α, 8α, 9α, 14α), α.α.α.β, α.α.β.α,α.β.α.α, β.α.α.α, α.α.β.β, α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α,α.β.β.β, β.α.β.β, β.β.α.β, β.β.β.α or β.β.β.β, typically α.α.β.α orβ.α.β.α.

3. The composition of embodiment 2 wherein the one or more formula 1compounds has the structure

wherein hydrogen atoms at the 5 (if present), 8, 9 and 14 positionsrespectively are α.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α, α.α.β.β,α.β.α.β, β.α.α.β, β.α.β.α, β.β.αα, α.β.β.α, α.β.β.β, β.α.β.β, β.β.α.β,β.β.β.α or β.β.β.β, typically α.α.β.α or β.α.β.α.

4. The composition of embodiments 1, 2 or 3 wherein one, two, three orfour formula 1 compounds are present.

5. The composition of embodiments 1, 2, 3 or 4 wherein the compositioncomprises less than about 0.3% v/v water.

6. The composition of embodiments 1, 2, 3, 4 or 5 wherein the one ormore nonaqueous liquid excipients is one, two or more of an alcohol, apolyethylene glycol, propylene glycol or benzyl benzoate.

7. The composition of any of embodiments 1-6 (embodiment 1, 2, 3, 4, 5or 6) wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7α-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7α,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one,3β,7α,-dihydroxyepiandrosterone, 3β,7β,-dihydroxyepiandrosterone,3β,-hydroxy-7-oxoepiandrosterone.

8. The composition of embodiment 7 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

9. The composition of any of embodiments 1-8 wherein the compositioncomprises two, three, four or five nonaqueous liquid excipients.

10. The composition of embodiment 9 wherein the composition comprisesthree or more nonaqueous liquid excipients.

11. The composition of any of embodiments 1-10 wherein the formula 1compound comprises about 0.0001-99% w/v of the composition.

12. The composition of any of embodiments 1-11 wherein the compositioncomprises a unit dose.

13. The composition of embodiment 12 wherein the unit dose comprisesabout 0.5-100 mg/mL of the formula 1 compound.

14. The composition of embodiment 10 wherein the composition comprisesabout 1.0-60 mg/mL of the formula 1 compound.

15. The composition of embodiment 14 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one or16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

16. The composition of embodiment 1 wherein the one or more nonaqueousliquid excipients comprise a polyethylene glycol, propylene glycol andbenzyl benzoate.

17. The composition of embodiment 16 wherein the composition comprisesless than about 0.3% v/v water.

18. The composition of embodiment 17 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one or16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

19. The composition of embodiment 18 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

20. The composition of embodiment 16 that further comprises an alcohol.

21. The composition of embodiment 20 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one or16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

22. The composition of embodiment 1 wherein the one or more nonaqueousliquid excipients comprise benzyl benzoate, a polyethylene glycol, analcohol and optionally an additional nonaqueous liquid excipient.

23. The composition of embodiment 22 wherein the composition comprisesless than about 0.3% v/v water.

24. The composition of embodiment 22 or 23 wherein the formula 1compound is 16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one or16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

25. The composition of embodiment 24 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

26. The composition of embodiment 22, 23, 24 or 25 wherein thepolyethylene glycol is polyethylene glycol 300 and/or polyethyleneglycol 200.

27. The composition of embodiment 26 wherein the alcohol is polyethyleneglycol is polyethylene glycol 300.

28. The composition of embodiments 22 or 23 that comprises about 2.5-25%v/v ethanol, about 1-10% v/v benzyl benzoate, about 10-35% v/vpolyethylene glycol 300, about 40-65% v/v propylene glycol and about2-60 mg/mL 16α-bromo-3β-hydroxy-5α-androstan-17-one.

28A. The composition of embodiments 22, 23, 24, 25 or 26 that comprisesabout 0.1-10% v/v benzyl benzoate, about 0.1-10% v/v benzyl alcohol,about 1-95% v/v polyethylene glycol 200, about 1-95% v/v propyleneglycol and about 2-60 mg/mL 16α-bromo-3β-hydroxy-5α-androstan-17-one.The embodiment 28A composition may comprise about 2% v/v benzylbenzoate, about 2% v/v benzyl alcohol, about 40% v/v polyethylene glycol200, about 51% v/v propylene glycol (qs) and about 50 mg/mL16α-bromo-3β-hydroxy-5α-androstan-17-one.

29. The composition of embodiment 28 that comprises about 12.5% v/vethanol, about 5% v/v benzyl benzoate, about 25% v/v polyethylene glycol300, about 57.5% v/v propylene glycol and about 50 mg/mL16α-bromo-3β-hydroxy-5α-androstan-17-one.

30. The composition of any of embodiments 1-29 that further comprises alocal anesthetic.

31. The composition of embodiment 30 wherein the local anesthetic isprocaine, benzocaine or lidocaine.

32. The composition of any of embodiments 1-31 wherein the compositioncomprises a solvate, a suspension, a colloid, a gel or a combination ofany of the foregoing.

33. A product produced by the process of contacting a compositioncomprising one or more compounds of formula 1 and a first nonaqueousliquid excipient with a second nonaqueous liquid excipient wherein theproduct comprises less than about 3% water and the salts, analogs,configurational isomers and tautomers thereof.

34. The product of embodiment 33 wherein the product comprises less thanabout 0.3% water.

35. The product of embodiments 33 or 34 wherein the first nonaqueousliquid excipient is a polyethylene glycol (e.g., PEG300 or PEG 200) orpropylene glycol.

36. The product of embodiments 33, 34 or 35 wherein the secondnonaqueous liquid excipient is a polyethylene glycol (e.g., PEG300 orPEG 200) or propylene glycol.

38. A product produced by the process of contacting a compositioncomprising one or more compounds of formula 1 and two nonaqueous liquidexcipients with a third nonaqueous liquid excipient wherein the productcomprises less than about 3% water and the salts, analogs,configurational isomers and tautomers thereof.

39. The product of embodiment 38 wherein the product comprises less thanabout 0.3% water.

40. The product of embodiments 38 or 39 wherein the two nonaqueousliquid excipients are selected from a polyethylene glycol (e.g., PEG300or PEG 200), propylene glycol, benzyl benzoate and an alcohol (e.g.,ethanol).

41. The product of embodiments 38, 39 or 40 wherein the third nonaqueousliquid excipient is a polyethylene glycol (e.g., PEG300 or PEG 200),propylene glycol, benzyl benzoate or an alcohol (e.g., ethanol).

42. A product produced by the process of contacting a compositioncomprising one or more compounds of formula 1 and three nonaqueousliquid excipients with a fourth nonaqueous liquid excipient wherein theproduct comprises less than about 3% water and the salts, analogs,configurational isomers and tautomers thereof.

43. The product of embodiment 42 wherein the product comprises less thanabout 0.3% water.

44. The product of embodiments 42 or 43 wherein the three nonaqueousliquid excipients are selected from a polyethylene glycol (e.g., PEG300or PEG 200), propylene glycol, benzyl benzoate and an alcohol (e.g.,ethanol).

45. The product of embodiments 42, 43 or 44 wherein the fourthnonaqueous liquid excipient is a polyethylene glycol (e.g., PEG300 orPEG 200), propylene glycol, benzyl benzoate or an alcohol (e.g.,ethanol).

46. The product of any of embodiments 33-45 wherein the product has beenstored at reduced temperature (about 4° C. to about 8° C.) or at ambienttemperature for about 30 minutes to about 2 years.

47. The product of any of embodiments 33-46 wherein the one or morecompounds of formula 1 comprise 1, 2, 3 or 4 formula 1 compounds.

48. The product of any of embodiments 33-46 wherein the one or morecompounds of formula 1 comprises one formula 1 compound.

49. The product of any of embodiments 33-48 wherein the one or moreformula 1 compound is selected from16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one and16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

50. The product of embodiment 49 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

51. The product of embodiment 49 that comprises about 2.5-25% v/vethanol, about 1-10% v/v benzyl benzoate, about 10-35% v/v polyethyleneglycol 300, about 40-65% v/v propylene glycol and about 2-60 mg/mL16α-bromo-3β-hydroxy-5α-androstan-17-one.

52. The product of embodiment 51 that comprises about 12.5% v/v ethanol,about 5% v/v benzyl benzoate, about 25% v/v polyethylene glycol 300,about 57.5% v/v propylene glycol and about 50 mg/mL16α-bromo-3β-hydroxy-5α-androstan-17-one.

53. The product of any of embodiments 33-52 that further comprises alocal anesthetic.

54. The composition of 52 wherein the local anesthetic is procaine,benzocaine or lidocaine.

55. A product produced by the process of contacting a compositioncomprising a compound of formula 1 with a nonaqueous liquid excipientwherein the product comprises less than about 3% v/v water and thesalts, analogs, configurational isomers and tautomers thereof.

56. The product of embodiment 55 wherein the product comprises less thanabout 0.3% v/v water.

57. The product of embodiment 53 wherein the product has been stored atreduced temperature (about 4° C. to about 8° C.) or at ambienttemperature for about 1 hour to about 2 years.

58. The product of embodiment 53 wherein the first nonaqueous liquidexcipient is a polyethylene glycol, an alcohol, propylene glycol orbenzyl benzoate.

59. The product of any of embodiments 33-58 wherein the formula 1compound comprises about 0.01% to about 99% w/v of the product.

60. The product of any of embodiments 33-59 wherein the product is aunit dose.

61. The unit dose of embodiment 60 comprising a solution containingabout 0.5-70 mg/mL of the one or more formula. 1 compound.

62. The product of any of embodiments 55-61 wherein the one or moreformula 1 compound is selected from16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5αandrostene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one and16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

63. The product of embodiment 62 wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

64. The product of any of embodiments 33-61 wherein the one or moreformula 1 compound is selected from the compounds or one or more of thespecies of compounds within the genera named in compound groups 1through 21-10-6.

65. A method comprising administering the composition or product of anyof embodiments 1-64 to a subject suffering from a pathogen infection ora malignancy or an immune suppression or disregulation condition, e.g.,a suppressed Th1 immune response or an unwanted Th2 immune response.

66. The method of embodiment 65 wherein the pathogen infection is a DNAvirus infection or an RNA virus infection.

67. The method of embodiment 66 wherein the RNA virus infection is aretrovirus infection or a hepatitis virus infection.

68. The method of embodiment 67 wherein the retrovirus infection orhepatitis virus infection is an HIV, FIV, SIV, SHIV or hepatitis C virusinfection.

69. The method of embodiment 65 wherein the pathogen infection is anintracellular parasite infection.

70. The method of embodiment 69 wherein the intracellular parasiteinfection is a malaria infection.

71. The method of embodiment 65 wherein the formula 1 compound has thestructure

wherein one, two or three of R⁷, R⁸ and R⁹ are —CH₂— or —CH═ and whereinthe configuration of hydrogen atoms at the 5 (if present), 8, 9 and 14positions respectively are α.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α,α.α.β.β, α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β,β.β.α.β, β. β.β.α or β.β.β.β, typically α.α.β.α or β.αβ.α.

72. The method of embodiment 71 wherein the formula 1 compound has thestructure

73. The method of embodiment 72 wherein R¹, R² and R⁴ independently are—OH, aC2-C20 ester or C1-C20 alkoxy, R³ is —H and two or three of R⁷, R⁸and R⁹ are —CH₂—.

74. The method of embodiment 72 or 73 wherein the formula I compound hasthe structure

75. The method of any of embodiments 71-74 wherein the configuration ofhydrogen atoms at the 5 (if present), 8, 9 and 14 positions respectivelyare α.α.β.α or β.α.β.α.

In other embodiments, the fomula 1 compounds include new compounds, someof which are described in the following numbered embodiments.

1A. A compound of formula 1 having the structure

wherein R⁷, R⁸ and R⁹ are independently selected and wherein one, two orthree of R⁷, R⁸ and R⁹ are not —CH₂— or —CH═ and wherein hydrogen atomsat the 5 (if present), 8, 9 and 14 positions respectively are in theα.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α, α.α.β.β, α.β.α.β, β.α.α.β,β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β, β.β.α.β, β.β.β.α or β.β.β.βconfigurations, typically α.α.β.α or β.α.β.α.

2A. The compound of embodiment 1A wherein R⁸ is —CH₂—, —O—, —S— or —NH—.

3A. The compound of embodiment 1A or 2A wherein R⁷ is —CH₂—CHR¹⁰—,—O—CHR¹⁰— or —O—C(O)—.

4A. The compound of embodiment 1A, 2A or 3A wherein R⁸ or R⁹ is absent.

5A. The compound of embodiment 1 A or 2A wherein R⁷ and R⁹ independentlyare —CHR¹⁰—, —CH₂—, —CH═, —O—, —S— or —NH—, wherein R¹⁰ is —OH, —SH, aC₁₋₃₀ organic moiety, a C₁₋₃₀ ester, C₁₋₁₀ optionally substituted alkyl,C₁₋₁₀ optionally substituted alkoxy, C₁₋₁₀ optionally substitutedalkenyl or C₁₋₁₀ optionally substituted alkynyl.

6A. The compound of embodiment 1A, 2A, 3A, 4A or 5A wherein the formula1 compound has the structure

wherein hydrogen atoms at the 5 (if present), 8, 9 and 14 positionsrespectively are in the α.α.β.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α,α.α.β.β, α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β,β.β.α.β, β.β.β.α or β.β.β.β configurations, typically α.α.β.α orβ.α.β.α.

7A. The compound of embodiment 6A wherein R⁴ is —OH, ═O, —SH, a C₁₋₃₀ester or C₁₋₃₀ alkoxy, wherein the ester or alkoxy moiety is optionallysubstituted with one, two or more independently selected substituents,which are optionally selected from —F, —Cl, —Br, —I, —O—, ═O, —S—, —NH—,—OR^(PR), —SR^(PR) or —NHR^(PR).

8A. The compound of embodiment 6A or 7A wherein R¹ is —OH, ═O, —SH, aC₁₋₃₀ ester or C₁₋₃₀ alkoxy, wherein the ester or alkoxy moiety isoptionally substituted with one, two or more independently selectedsubstituents, which are optionally selected from —F, —Cl, —Br, —I, —O—,═O, —S—, —NH—, —OR^(PR), —SR^(PR) or —NHR^(PR),

9A. The compound of embodiment 1A, 2A or 3A wherein the formula 1compound has the structure

wherein hydrogen atoms at the 5 (if present), 8, 9 and 14 positionsrespectively are α.α.α.α, α.α.α.β, α.α.β.α, α.β.α.α, β.α.α.α, α.α.β.β,α.β.α.β, β.α.α.β, β.α.β.α, β.β.α.α, α.β.β.α, α.β.β.β, β.α.β.β, β.β.α.β,β.β.β.α, or β.β.β.β, typically α.α.β.α or β.α.β.α.

10A. The compound of embodiment 9A wherein R⁴ is —OH, ═O, —SH, a C₁₋₃₀ester or C₁₋₃₀ alkoxy, wherein the ester or alkoxy moiety is optionallysubstituted with one, two or more independently selected substituents,which are optionally selected from —F, —Cl, —Br, —I, —O—, ═O, —S—, —NH—,—OR^(PR), —SR^(PR) or —NHR^(PR).

11A. The compound of embodiment 9A or 10A wherein R¹ is —OH, ═O, —SH, aC₁₋₃₀ ester or C¹⁻³⁰ alkoxy, wherein the ester or alkoxy moiety isoptionally substituted with one, two or more independently selectedsubstituents, which are optionally selected from —F, —Cl, —Br, —I, —O—,═O, —S—, —NH—, —OR^(PR), —SR^(PR) or NHR^(PR)

12A. A composition comprising a compound of any of embodiments 1A-11Aand an excipient suitable for human pharmaceutical use or for veterinaryuse, e.g., an excipient disclosed herein or in the cited references.

13A. A product produced by the process of contacting a compound of anyof embodiments 1A-11A and an excipient suitable for human pharmaceuticaluse or for veterinary use, e.g., an excipient disclosed herein or in thecited references.

14A. The use of a compound, composition or product of any of embodimentsembodiments 1A-13A to prepare a medicament for use to prevent or totreat, or to ameliorate one or more symptoms associated, with aninfection, an immunesuppression condition, a malignancy, a pre-malignantcondition or to modulate a mammal's immune response, such as enhancing aTh1 response or decreasing a Th2 response, e.g., an infection,malignancy or immune dysregulation as described herein or in the citedreferences.

15A. The use of embodiment 14A, wherein the infection is a viralinfection (e.g., HIV, HCV, a Herpesvirus, a togavirus, a human papillomavirus infection or other virus described herein or in the citedreferences), a bacterial infection (e.g., Borrelia sp., Legionella sp.or other bacterium described herein or in the cited references), afungal or a yeast infection (e.g., Candida sp., Aspergillus sp. or otheryeast described herein or in the cited references) or a parasiteinfection (e.g., a malaria parasite, a gastrointestinal nematode, ahelminth, Leishmania sp., Cryptosporidium sp., Toxoplasma gondii,Pneumocystis carinii, Schistosoma sp., Strongyloides stercoralis orother parasite described herein or in the cited references).

16A. The compound, composition, product or use of any of embodiments1A-15A, wherein the formula 1 compound is a compound named in any ofcompound groups 1 through 42-25-10-6, or the formula 1 compound is aspecies in any genus described in any of compound groups 1 through42-25-10-6.

In other aspects, the invention provides dosing methods suitable totreat the conditions described herein. The following embodimentsdescribe some of these methods.

1B. A method comprising intermittently administering one or morecompounds of formula 1 (or a composition comprising a formula 1compound) to a subject or delivering to the subject's tissues a formula1 compound(s) (or a composition comprising a formula 1 compound), e.g.,any formula 1 compound named or described herein, including thecompounds described in embodiments 1-64 and 1A-11A above.

2B. The method of embodiment 1B wherein the subject has an infection, ahyperproliferation disorder, a hypoproliferation condition, animmunosuppression condition, an unwanted immune response or wherein thesubject has recently experienced or will shortly experience trauma,surgery or a therapeutic treatment wherein the therapeutic treatment isone other than the method of embodiment 1B.

3B. The method of embodiment 2B wherein the immunosuppression conditionor the unwanted immune response is associated with a viral infection, anintracellular bacterial infection, an extracellular bacterial infection,a fungal infection, a yeast infection, an extracellular parasiteinfection, an intracellular parasite infection, a protozoan parasite, amulticellular parasite, an autoimmune disease, a cancer, a precancer, achemotherapy, a radiation therapy, an immunosuppressive therapy, ananti-infective agent therapy, a wound, a burn, the presence of animmunosuppressive molecule, gastrointestinal irritation, or anycombination of the foregoing.

4B. The method of embodiment 3B wherein the subject's immunosuppressioncondition is ameliorated or the unwanted immune response (e.g., a Th2response) is reduced or wherein the subject's Th1 immune response isenhanced.

5B. The method of embodiment 3B wherein the subject's innate immunity,specific immunity or both is enhanced.

6B. The method of embodiment 5B wherein the subject's innate immunity isenhanced.

7B. The method of embodiment 6B wherein the subject's specific immunityis enhanced, e.g., wherein the subject's Th2 immune response is reducedor wherein the subject's Th1 immune response is enhanced.

8B. The method of embodiment 2B wherein the one or more compounds offormula 1 is or are administered according to the a dosing regimencomprising the steps,

(a) administering the one or more compounds of formula 1 to the subjectat least once per day for at least 2 days;

(b) not administering the one or more formula 1 compounds to the subjectfor at least 1 day;

(c) administering the one or more formula 1 compounds to the subject atleast once per day for at least 2 days; and

(d) optionally repeating steps (a), (b) and (c) at least once orvariations of steps (a), (b) and (c) at least once.

9B. The method of embodiment 8B wherein step (c) comprises the samedosing regimen as step (a).

10B. The method of embodiment 9B wherein step (a) of the dosing regimencomprises administering the one or more formula 1 compounds once perday, twice per day, three times per day or four times per day.

11B. The method of embodiment 10B wherein step (a) of the dosing regimencomprises administering the one or more formula 1 compounds once per dayor twice per day.

12B. The method of embodiment 10B wherein step (a) comprisesadministering the one or more formula 1 compounds for about 3 to about24 days.

13B. The method of embodiment 12B wherein step (a) comprisesadministering the one or more formula 1 compounds for about 4 to about12 days.

14B. The method of embodiment 13B wherein step (a) comprisesadministering the one or more formula 1 compounds for about 4 to about 8days.

15B. The method of embodiment 14B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 3 to about120 days.

16B. The method of embodiment 15B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 4 to about60 days.

17B. The method of embodiment 16B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 5 to about30 days.

18B. The method of embodiment 16B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 8 to about60 days.

19B. The method of embodiment 15B wherein steps (a), (b), and (c) arerepeated at least about 4 times.

20B. The method of embodiment 15B wherein steps (a), (b), and (c) arerepeated about 5 times to about 25 times.

21B. The method of embodiment 15B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 2 months.

22B. The method of embodiment 15B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 12 months.

23B. The method of embodiment 8B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 3 to about120 days.

24B. The method of embodiment 23B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 4 to about60 days.

25B. The method of embodiment 24B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 5 to about30 days.

26B. The method of embodiment 23B wherein step (b) comprises notadministering the one or more formula 1 compounds for about 8 to about60 days.

27B. The method of embodiment 8B wherein step (d) comprises repeatingsteps (a), (b), and (c) at least once.

28B. The method of embodiment 27B wherein step (d) comprises repeatingsteps (a), (b), and (c) about 3 times to about 25 times.

29B. The method of embodiment 1B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 2 months.

30B. The method of embodiment 29B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 12 months.

31B. The method of any of embodiments 8B-30B wherein theimmunosuppression condition or the unwanted immune response isassociated with a viral infection, an intracellular bacterial infection,an extracellular bacterial infection, a fungal infection, a yeastinfection, an extracellular parasite infection, an intracellularparasite infection, a protozoan parasite, a multicellular parasite, anautoimmune disease, a cancer, a precancer, a chemotherapy, a radiationtherapy, an immunosuppressive therapy, an anti-infective agent therapy,a wound, a burn, the presence of an immunosuppressive molecule,gastrointestinal irritation or any combination of the foregoing.

32B. The method of embodiment 31B wherein the subject'simmunosuppression condition is ameliorated or the unwanted immuneresponse is reduced.

33B. The method of embodiment 32B wherein the subject's innate immunity,specific immunity or both is enhanced.

34B. The method of embodiment 33B wherein the subject's innate immunityis enhanced.

35B. The method of embodiment 34B wherein the subject's specificimmunity is enhanced.

36B. The method of embodiment 8B wherein step (c) comprises the ashorter dosing regimen than step (a).

37B. The method of embodiment 36B wherein step (a) comprisesadministering the formula 1 compound for 7 to about 24 days.

38B. The method of embodiment 37B wherein step (c) comprisesadministering the formula 1 compound for 4 to about 12 days.

39B. The method of embodiment 38B wherein step (b) comprises notadministering the formula 1 compound for about 3 to about 120 days.

40B. The method of embodiment 39B wherein step (b) comprises notadministering the formula 1 compound for about 4 to about 60 days.

41B. The method of embodiment 40B wherein step (b) comprises notadministering the formula 1 compound for about 5 to about 30 days.

42B. The method of embodiment 36B wherein step (d) comprises repeatingsteps (a), (b), and (c) at least once.

43B. The method of embodiment 42B wherein step (d) comprises repeatingsteps (a), (b), and (c) about 3 times to about 25 times.

44B. The method of embodiment 36B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 2 months.

45B. The method of embodiment 44B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 12 months.

46B. The method of any of embodiments 36B-45B wherein theimmunosuppression condition or the unwanted immune response isassociated with a viral infection, an intracellular bacterial infection,an extracellular bacterial infection, a fungal infection, a yeastinfection, an extracellular parasite infection, an intracellularparasite infection, a protozoan parasite, a multicellular parasite, anautoimmune disease, a cancer, a precancer, a chemotherapy, a radiationtherapy, an immunosuppressive therapy, an anti-infective agent therapy,a wound, a burn, the presence of an immunosuppressive molecule,gastrointestinal irritation or any combination of the foregoing.

47B. The method of embodiment 46B wherein the subject'simmunosuppression condition is ameliorated or the unwanted immuneresponse is reduced.

48B. The method of embodiment 47B wherein the subject's innate immunity,specific immunity or both is enhanced.

49B. The method of embodiment 48B wherein the subject's innate immunityis enhanced.

50B. The method of embodiment 48B wherein the subject's specificimmunity is enhanced.

51B. The method of embodiment 8B wherein step (c) comprises a longerdosing period than step (a).

52B. The method of embodiment 51B wherein step (a) comprisesadministering the formula 1 compound for 7 to about 24 days.

53B. The method of embodiment 52B wherein step (c) comprisesadministering the formula 1 compound for 4 to about 12 days.

54B. The method of embodiment 53B wherein step (b) comprises notadministering the formula 1 compound for about 3 to about 120 days.

55B. The method of embodiment 54B wherein step (b) comprises notadministering the formula 1 compound for about 4 to about 60 days.

56B. The method of embodiment 55B wherein step (b) comprises notadministering the formula 1 compound for about 5 to about 30 days.

57B. The method of embodiment 51B wherein step (d) comprises repeatingsteps (a), (b), and (c) at least once.

58B. The method of embodiment 57B wherein step (d) comprises repeatingsteps (a), (b), and (c) about 3 times to about 25 times.

59B. The method of embodiment 51B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 2 months.

60B. The method of embodiment 59B wherein steps (a), (b), and (c) andrepetitions of steps (a), (b), and (c) occur over a time period of atleast about 12 months.

61B. The method of any of embodiments 51B-60B wherein theimmunosuppression condition or the unwanted immune response isassociated with a viral infection, an intracellular bacterial infection,an extracellular bacterial infection, a fungal infection, a yeastinfection, an extracellular parasite infection, an intracellularparasite infection, a protozoan parasite, a multicellular parasite, anautoimmune disease, a cancer, a precancer, a chemotherapy, a radiationtherapy, an immunosuppressive therapy, an anti-infective agent therapy,a wound, a burn, the presence of an immunosuppressive molecule,gastrointestinal irritation or any combination of the foregoing.

62B. The method of embodiment 61B wherein the subject'simmunosuppression condition is ameliorated or the unwanted immuneresponse is reduced.

63B. The method of embodiment 62B wherein the subject's innate immunity,specific immunity or both is enhanced or wherein the subject's Th1immune response is enhanced or the subject's Th2 immune response isdecreased.

64B. The method of embodiment 8B wherein the variations of steps (a),(b) and (c) comprise conducting a first dosing regimen of steps (a), (b)and (c) once, twice or three times, followed by one or more seconddosing regimens of steps (a′), (b′) and (c′) wherein one or more of the(a′), (b′) and (c′) steps in the second dosing regimen is longer thanthe corresponding step in the first dosing regimen.

65B. The method of embodiment 8B wherein the variations of steps (a),(b) and (c) comprise conducting a first dosing regimen of steps (a), (b)and (c) once, twice or three times, followed by one or more seconddosing regimens of steps (a′), (b′) and (c′) wherein one or more of the(a′), (b′) and (c′) steps in the second dosing regimen is shorter thanthe corresponding step in the first dosing regimen.

66B. The method of any of embodiments 1B-67B wherein the one or moreformula 1 compounds is or are administered orally, intramuscularly,intravenously, subcutaneously, topically, vaginally, rectally,intracranially, intrathecally, intradermally, as an aerosol or by abuccal route.

67B. The method of embodiment 66B wherein the one or more formula 1compounds is or are present in a solid formulation predominantly as asolid or the one or more formula 1 compounds is or are present in aliquid formulation predominantly as a solvate, colloid or a suspensionor the one or more formula 1 compounds is or are present in a gel, creamor paste.

68B. The method of any of embodiments 2B-67B wherein the subject's viralinfection, intracellular bacterial infection, extracellular bacterialinfection, fungal infection, yeast infection, extracellular parasiteinfection, intracellular parasite infection, protozoan parasite,multicellular parasite, autoimmune disease, cancer, precancer,chemotherapy, radiation therapy, immunosuppressive therapy,anti-infective agent therapy, a wound, a burn, or the presence of animmunosuppressive molecule, gastrointestinal irritation or anycombination of the foregoing is (a) a DNA virus infection or an RNAvirus infection (HSV, CMV, HBV, HCV, HIV, SHIV, SIV); (b) a mycoplasmainfection, a Listeria infection or a Mycobacterium infection; (c)extracellular bacteria infection; (d) fungal infection; (e) a yeastinfection (Candida, Cryptococcus); (d) protozoa (malaria, leishmania,cryptosporidium, toxoplasmosis, pneumocystis); (e) a multicellularparasite; (f) autoimmune diseases (SLE, RA, diabetes); (g) cancers(solid cancers selected from, e.g., ovarian, breast, prostate, glioma;disseminated cancers selected from lymphoma, leukemia, colon cancer,sarcoma); (h) precancers; (i) chemotherapies (adriamycin, cisplatin,mitomycin C); o) radiation therapies; (k) immunosuppressive therapies;(l) anti-infective agent therapies; (m) wounds (surgical or otherwise);(n)1^(st) degree, 2^(nd) degree or 3^(rd) degree burns; (o)immunosuppressive molecules; (p) gastrointestinal irritation (irritablebowel, Crohn's disease, chronic diarrhea); or (q) any combination of (a)through (p).

69B. The method of embodiment 68B wherein the RNA virus infection is aretroviral infection or a hepatitis virus infection.

70B. The method of embodiment 68B or 69B wherein the one or more formula1 compounds is one formula 1 compound.

71B. The method of embodiment 70B wherein the one or more formula 1compounds is or are in a composition that comprises, (a) one or morenonaqueous liquid excipients, wherein the composition comprises lessthan about 3% v/v water; (b) a solid that comprises a pharmaceuticallyacceptable excipient; or (c) one or more liquid excipients, wherein thecomposition comprises more than about 3% v/v water.

72B. The method of embodiment 68B or 71B wherein the formula 1 compoundis 16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one or16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one.

73B. The method of embodiment 72B wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one.

74B. The method of embodiments 1B-73B wherein the formula 1 compoundexcludes one or more of any formula 1 compounds.

75B. A method to treat involuntary weight loss, oral lesions, skinlesions, opportunistic infections, diarrhea or fatigue in an subjectcomprising intermittently administering one or more compounds of formula1 to the subject (e.g., involuntary weight loss from viral infection,gastrointestinal infection, chemotherapy, anorexia).

76B. The method of embodiment 75B wherein the subject has animmunosuppression condition.

77B. The method of embodiment 76B wherein the subject is a human.

78B. The method of embodiment 77B wherein the subject is a human 1 dayto 18 years old (e.g., 1 month to 6 years old).

79B. The method of any of embodiments 75B-78B wherein the subject'sspecific immunity remains impaired compared to a typical comparablecontrol subject who does not have the subject's pathological condition.

80B. The method of embodiment 79B wherein the subject's CD4 cell countdoes not increase significantly during one or more courses of dosing(e.g., dosing for 1 week to about 2 weeks or more).

81B. The method of clam 80B wherein the subject's CD4 cell count isabout 20 to about 100 CD4⁺ cells/mm³ or about 20 to about 75 CD4⁺cells/mm³.

82B. The method of any of embodiments 1B-81B wherein the subject has apathogen(s) infection or a malignancy and the pathogen(s) or malignancydoes not become resistant to the formula 1 compound over a time normallyassociated with the development of measurable resistance in at leastabout 50% of subjects who are treated with a therapeutic treatment(s)other than a formula 1 compound(s).

83B. The method of embodiment 82B wherein the pathogen infection is anHIV, SIV, SHIV or HCV infection.

84B. The method of embodiments 82B or 83B wherein the formula 1 compoundis one or more of 16α-bromo-3β-hydroxy-5α-androstan-17-one,16α-bromo-3β,7β-dihydroxy-5α-androstan-17-one,16α-bromo-3β,7β,17β-trihydroxy-5α-androstene,16α-bromo-3β,7β-dihydroxy-5α-androstane,16α-bromo-3β,7β-dihydroxy-5α-androstene,16α-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstane,16β-bromo-3β,17β-dihydroxy-5α-androstene,16β-bromo-3β,7β,17β-trihydroxy-5α-androstane,16β-bromo-3β-hydroxy-5α-androstan-17-one,16β-bromo-3β-hydroxy-5α-androsten-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androstan-17-one,16β-bromo-3β,7β,-dihydroxy-5α-androsten-17-one or a physiologicallyacceptable ester, carbonate, carbamate, amino acid conjugate or peptideconjugate thereof.

85B. The method of embodiment 84B wherein the formula 1 compound is16α-bromo-3β-hydroxy-5α-androstan-17-one or a physiologically acceptableester, carbonate, carbamate, amino acid conjugate or peptide conjugatethereof.

86B. The method of any of embodiments 1B-85B, wherein the formula 1compound is a compound named in any of compound groups 1 through42-25-10-6, or the formula 1 compound is a species in any genusdescribed in any of compound groups 1 through 42-25-10-6.

In other embodiments, the invention provides methods to modulate immunecells or immune responses in a subject. The following numberedembodiments describe some of these methods.

1C. A method to modulate a subject's innate immunity or to enhance asubject's Th1 immune response or to reduce a subject's Th2 immuneresponses comprising administering to the subject a compound(s) offormula 1, including any formula 1 compound that is described ordisclosed herein, including the compounds described in embodiments 1-64and 1A-11A above.

2C. The method of embodiment 1C wherein the subject's innate immunity isenhanced.

3C. The method of embodiment 1C or 2C wherein the subject suffers froman innate immunity suppression condition, a suppressed Th1 immuneresponse or an unwanted Th2 immune response.

4C. The method of embodiment 3C wherein the innate immunity suppressioncondition, the suppressed Th1 immune response or the unwanted Th2response is associated with a viral infection, an intracellularbacterial infection, an extracellular bacterial infection, a fungalinfection, a yeast infection, an extracellular parasite infection, anintracellular parasite infection, a protozoan parasite, a multicellularparasite, an autoimmune disease, a cancer, a precancer, a chemotherapy,a radiation therapy, an immunosuppressive therapy, an anti-infectiveagent therapy, a wound, a burn, the presence of an immunosuppressivemolecule or any combination of the foregoing.

5C. The method of any of embodiments 1C-3C wherein the subject's Th1immune response is enhanced.

6C. The method of embodiment 1C wherein the subject's Th2 immuneresponse is reduced.

7C. The method of embodiment 6C wherein the subject has a conditioncomprising an unwanted immune response (e.g., autoimmune disease, SLE,diabetes).

9C. The method of embodiment 6C or 7C wherein the subject is avertebrate, a mammal, a primate or a human.

10C. The method of embodiment 9 wherein the vertebrate's, the mammal'sthe primate's or the human's specific immunity modulation is (i) anenhanced CTL or Th1 response to a virus infection or to a malignant cellin vitro or in vivo, (ii) enhanced antigen presentation or biologicalactivity by dendritic cells or dendritic cell precursors, or (iii)enhanced killing of virus-infected or of malignant cells.

11C. The method of 10C. wherein the vertebrate is a human, the virusinfection is an HIV infection and the CTL or Th1 response comprises anenhanced response to one or more of the HIV's gag protein or to theHIV's gp120.

12C. The method of embodiment 1C, 4C, 10C or 11C wherein the subject'sTh1 cells, tumor-infiltrating lymphocytes (TIL cells), NK cells,peripheral blood lymphocytes, phagocytes, monocytes, macrophage,neutrophils, eosinophils, dendritic cells or fibrocytes are activated asmeasured by, e.g., enhanced ³H-thymidine uptake compared to untreatedcontrols or by an increase in the number of the cell type in circulationor demonstrable movement of the cell type from one tissue or compartment(e.g., skin) to another tissue or compartment (e.g., blood, lymph node,spleen or thymus).

13C. The method of embodiment 1C, 4C, 10C, 11C or 12C, wherein theformula 1 compound(s) modulates transcription of one or more genes inthe subject's NK cells, phagocytes, monocytes, macrophages, neutrophils,eosinophils, dendritic cells or fibrocytes are activated (e.g., asmeasured by increased protein kinase C activity or by modulation of abiological activity of a steroid receptor or an orphan nuclear hormonereceptor).

14C. The method of embodiment 1C wherein the formula 1 compound(s)enhances lysosome movement in one or more of the subject's NK cells,phagocytes, monocytes, macrophages, neutrophils, eosinophils, dendriticcells or fibrocytes.

15C. The method of embodiment 1C wherein the formula 1 compound(s)enhances protein kinase C activity in one or more of the subject's NKcells, phagocytes, monocytes, macrophages, neutrophils, eosinophils,dendritic cells or fibrocytes (e.g., PKCα, PKCβ, PKCγ and PKCζ).

16C. A composition comprising a partially purified or a purified complexcomprising a formula 1 compound and a steroid receptor, a serumsteroid-binding protein (e.g., human serum albumin, α1-acidglycoprotein, sex hormone-binding globulin, testosterone-bindingglobulin, corticosteroid-binding globulin, androgen binding protein(rat)) or a binding partner (e.g., complexing agent, liposome,antibody).

17C. A product produced by the process of contacting the partiallypurified or the purified composition of embodiment 16C with one or moresterile containers, one or more syringes, one or more pharmaceuticallyacceptable excipients (e.g., excipient as defined in specification aboveand including sugars, lactose, sucrose, fillers, lubricants, binders, orany excipient named in any reference cited herein), one or more cells,one or more tissues, plasma or blood.

18C. The method of any of embodiments 1C-17C wherein the subject has aninfection, a hyperproliferation disorder, a hypoproliferation condition,an immunosuppression condition, an unwanted immune response or whereinthe subject has recently experienced or will shortly experience trauma,surgery or a therapeutic treatment wherein the therapeutic treatment isone other than the method of embodiment 1C.

19C. The method of embodiment 18C wherein the immunosuppressioncondition or the unwanted immune response is associated with a viralinfection, an intracellular bacterial infection, an extracellularbacterial infection, a fungal infection, a yeast infection, anextracellular parasite infection, an intracellular parasite infection, aprotozoan parasite, a multicellular parasite, an autoimmune disease, acancer, a precancer, a chemotherapy, a radiation therapy, animmunosuppressive therapy, an anti-infective agent therapy, a wound, aburn, the presence of an immunosuppressive molecule, gastrointestinalirritation, or any combination of the foregoing.

20C. The method of embodiment 19C wherein the subject'simmunosuppression condition is ameliorated or the unwanted immuneresponse is reduced.

21C. The method of embodiment 19C wherein the subject'simmunosuppression condition is associated with a viral infection.

22C. The method of embodiment 21C wherein the viral infection comprisesa DNA virus or an RNA virus infection.

23C. The method of embodiment 22C wherein the RNA virus infectioncomprises a retroviral infection or a hepatitis virus infection.

24C. The method of any of embodiments 18C-23C wherein the subjectsuffers from one or more of chronic diarrhea, involuntary weight loss(usually at least about 5% or more), cachexia (usually at least about 5%or more), muscle wasting, one or more oral lesions (usually at leastabout 1 cm²), one or more genital lesions (usually at least about 1cm²), skin lesions (usually at least about 1 cm²) or an opportunisticinfection associated with AIDS.

25C. A method (e.g., to determine a biological activity of a formula 1compound or to modulate transcription of a gene in a cell or cell-freetranscription system) comprising: (a) contacting the formula 1compound(s) with a cell or cell population in vitro or in vivo; (b)measuring one or more of (i) a complex between a binding partner and theformula 1 compound, (ii) proliferation of the cell or cell population,(iii) differentiation of the cell or cell population (iv) an activity ofa protein kinase C, (v) a level of phosphorylation of a protein kinase Csubstrate, (vi) transcription of one or more target genes, (vii)enhancement or inhibition of the cellular response to steroids, e.g.,glucocorticoids, (viii) inhibition of steroid-induced transcription,e.g., glucocorticoids, sex steroids, (ix) inhibition of retrovirus(e.g., HIV, SIV, FIV or SHIV) LTR-driven transcription, or (x)modulation of the numbers of an immune cell population in circulation invivo (e.g., circulating peripheral blood lymphocytes in a mammal such asa primate or a human); and (c) optionally comparing the result obtainedin step (b) with an appropriate control.

26C. The method of embodiment 25C wherein the binding partner is asteroid receptor, a transcription factor or a steroid hormonesuperfamily orphan receptor.

27C. The method of embodiment 25C wherein the biological activitydetermined is a modulating activity of the formula 1 compound forreplication or cytopathic effects associated with a retrovirus, ahepatitis virus or a protozoan parasite.

28C. The method of embodiment 25C wherein the biological activitydetermined is a modulating activity of the formula 1 compound forreplication, cytopathic effects associated with the retrovirus, thehepatitis virus or the protozoan parasite or the biological activitydetermined is metabolism (assay by ³H-thymidine uptake) of a cell orcell population comprising NK cells, phagocytes, monocytes, macrophages,basophils, eosinophils, fibrocytes, transformed cells, virus-infectedcells, bacteria-infected cells or parasite-infected cells.

29C. The method of embodiment 25C wherein the target gene is a virusgene, a bacterial gene, a parasite gene, a gene associated with cancer.

30C. The method of embodiment 29C wherein the virus gene is a polymerasegene, a reverse transcriptase gene, an envelope gene, a protease gene ora gene associated with viral nucleic acid replication or a viralstructural gene.

31C. The method of embodiment 30C wherein the polymerase gene encodes aDNA polymerase or encodes an RNA polymerase.

32C. The method of embodiment 30C wherein the reverse transcriptase geneencodes a human, primate, avian or feline retrovirus reversetranscriptase.

33C. A method comprising administering a compound(s) of formula 1 to ahuman or primate who has a retroviral infection and a CD4 count of 550or less.

34C. The method of embodiment 33C wherein the human has a CD4 count ofabout 20 to about 100 or about 20 to about 80.

35C. The method of embodiment 33C wherein the human has a CD4 count ofabout 30 to about 150.

36C. The method of embodiment 33C wherein the human has a CD4 count ofabout 500 or less, about 450 or less, about 400 or less, about 350 orless, about 300 or less, about 250 or less, about 200 or less, about 150or less, about 100 or less, about 50 or less or about 25 or less orabout 20 or less.

37C. The method of any of embodiments 33C-36C wherein the formula 1compound(s) is present in a composition that comprises one or morenonaqueous liquid excipients and less than about 3% v/v water or any ofthe formulations as disclosed in the specification or any of thenumbered embodiments above.

38C. The method of any of embodiments 33C-37C wherein the formula 1compound(s) is administered according to an intermittent dosing protocolas disclosed in the specification or any of the numbered embodimentsabove.

39C. The method of any of embodiments 30C-45C wherein the human iscoinfected with hepatitis C virus, hepatitis B virus, HSV-1, HSV-2, amalaria parasite, a Pneumocystis parasite, or a Cryptosporidiumparasite.

40C. The method of embodiment 46C wherein level of the HCV is reduced inthe human.

41C. A method comprising administering a formula 1 compound(s) to asubject, or to a nervous system cell(s) in tissue culture whereby theformula 1 compound(s) binds to a receptor associated with a cell(s) inthe nervous system and (1) elicits a biological response in the cell(s)in the nervous system or in the cell(s) in tissue culture and/or (2)elicits a neuronal response that is transmitted to a distant site(s) orcell(s) where the method optionally is used to screen a formula 1compound(s) for its biological activity, to treat a pathologicalcondition (e.g., pathogen infection such as a virus (HIV), a malignancyor a neurological disorder, e.g., AIDS associated dementia, Alzheimer's,Parkinson's, Multiple Sclerosis) in the subject or to determine thebioavailability or metabolism of the formula 1 compound(s) to thesubject or the cell(s) in the nervous system or in tissue culture,wherein metabolism is optionally determined by comparing the biologicaleffect of a formula 1 compound(s) with a control compound, which can bea different formula 1 compound.

42C. The method of embodiment 41 wherein the receptor associated withthe cell in the nervous system is a neurotransmitter receptor(s) (e.g.,a γ-aminobutyric acid receptor such as type A, a NMDA receptor) and/or asteroid receptor (e.g., androgen receptor, estrogen receptor).

43C. The method of embodiment 41C or 42C wherein the cell(s) in thenervous system is a neuron(s), and astrocyte(s) and/or a glial cell(s).

44C. The method of embodiment 41C, 42C or 43C wherein the biologicalresponse in the cell(s) in the nervous system or in the cell(s) intissue culture is increased or decreased transcription of a gene(s)(e.g., a neurotransmitter, vasopressin, a heat shock protein), increasedor decreased secretion of a protein(s) (e.g., vasopressin), reduceddamage from oxidative stress, enhanced nitric oxide release and/orenhanced neurite growth.

45C. The method of any of embodiments 1C-44C wherein the compound(s) offormula 1 is any one or more formula 1 compound selected from thecompounds or one or more of the species of compounds within the generanamed in compound groups 1 through 21-10-6.

46C. A method to (a) modulate the expression of at least one immune cellantigen by an immune cell in a subject, wherein the immune cell antigenis selected from CD3, CD11c, CD14, CD16, CD19, CD25, CD38, CD56, CD62L,CD69, CD45RA, CD45RO, CD123, HLA-DR, IL-1, IL-2, IL-4, IL-6, IL-8,IL-10, TNFα, IGF₁ and γIFN, or (b) activate CD8⁺ T cells or CD8⁻ T cellsin a subject, wherein the activation comprises at least transientlyenhanced expression of CD25 or CD69 by the T cells, or (c) increase theproportion of CD8⁺ or CD8⁻ lymphokine activated killer cells in asubject's CD16+ cells (e.g., CD8⁺, CD16⁺, CD38⁺ or cells CD8⁻, CD16⁺,CD38⁺), or (d) increase the proportion of (i) CD8⁻, CD16⁺ natural killercells, (ii) CD8⁺, CD16⁺ natural killer cells or (iii) CD8⁻, CD16⁺ cellsthat mediate antibody-dependent cell-mediated cytotoxicity, or (iv)CD8⁺, CD16⁺ cells that mediate antibody-dependent cell-mediatedcytotoxicity, or (e) increase the proportion of dendritic cellprecursors in a subject's circulating white blood cells (e.g., Lin⁻,HLA-DR⁺, CD123⁺ or Lin⁻ HLA-DR⁺, CD11c⁺ cells) or (f) increase theproportion of CD45RA⁺ T cells or CD45⁺, R0⁺ T cells in a subject'scirculating white blood cells, or (g) change (increase or decrease) theproportion or relative numbers of CD62L⁺ T cells in a subject'scirculating white blood cells, or (h) increase the proportion of CD8⁺ orCD4⁺ T cells that express CD62L in a subject's circulating CD8⁺ or CD4⁺T cells, or (i) decrease the proportion of CD8⁺ or CD4⁺ T cells thatexpress CD62L in a subject's circulating CD8⁺ or CD4⁺ T cells, or (j)increase the proportion of HLA-DR⁺, CD8⁺, CD38₊ cells in a subject'scirculating white blood cells, or (k) decrease the level of IL-4 orIL-10 that is expressed by or present in a subject's white blood cellsor in a subject's plasma (or that is expressed after the subject's whitecells are stimulated in vitro), (l) at least transiently increase thenumber of dendritic cell precursors or dendritic cells that are presentin a subject's white blood cells or in a subject's plasma, or (m)enhance the capacity of CD4⁺ T cells to express IL-2, IL-12 or γIFN, themethod comprising administering to the subject a formula 1 compound anda pharmaceutically acceptable excipient.

47C. The method of embodiment 46C wherein formula 1 has the structure

wherein R¹ is —OH or a group (e.g., a C1-30 ester) that canhydrolytically convert under physiological conditions to —OH, either ofwhich may be in the α- or β-configuration; R² is hydrogen in the α- orβ-configuration, or R² is absent when there is a double bond at the 5-6position; R³ is —H or —Br, either of which may be in the α- orβ-configuration; R⁴ is —OH or a group (e.g., a C1-30 ester) that canhydrolytically convert under physiological conditions to —OH, either ofwhich may be in the α- or β-configuration, or R⁴ is ═O and the hydrogenatom bonded to the same carbon is absent; R^(4A) is R⁴, —C(O)—CH³ or—C(O)—(CH₂)₁₋₆—CH³; R⁵ is —H or —OH or a group (e.g., a C1-30 ester)that hydrolytically convert under physiological conditions to —OH,either of which may be in the α- or β-configuration, or R⁵ is ═O and thehydrogen atom bonded to the same carbon is absent; and the dotted lineat the 5-6 position is an optional double bond, or wherein the formula 1compound has the structure shown in any formula 1 compound named ordescribed herein, including the compounds described in embodiments 1-64and 1A-11A above.

48C. The method of embodiment 46C or 47C wherein the formula 1 compoundis administered to the subject daily over a period from one to about 15days, e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ormore days.

49C. The method of embodiment 48C wherein the expression of the immunecell antigen is detectably modulated for at least about 4-7 days afterthe last administration of the formula 1 compound to the subject, e.g.,for at least 4, 5, 6, 7 or more days.

50C. The method of embodiment 48C or 49C wherein the expression of theimmune cell antigen is detectable at least about 8-90 days after thelast administration of the formula 1 compound, e.g., for at least about8,10, 12, 15, 20, 25, 28, 30, 35, 40, 42, 45, 49, 50, 55, 56, 60, 63,65, 70, 75, 77, 80, 84, 85, 90, 91 95, 98, 100 or more days.

51C. The method of any of embodiments 46C-51C wherein the subject has animmunosuppression condition, a pathogen infection or a conditionsassociated with a deficient Th1 immune response or an excessive Th2immune response.

52C. The method of embodiment 51C wherein the pathogen infection is aviral infection, a bacterial infection, a yeast infection, a fungalinfection or a viroid infection, e.g., wherein the pathogen infection isa viral infection such as a DNA virus infection or an RNA virusinfection (e.g., an infection caused by a Hepadnavirus, a Parvovirus, aPapovavirus, an Adenovirus, a Herpesvirus, Retrovirus, a Flavivirus, aTogavirus, a Rhabdovirus, a Picornavirus, a Bunyavirus, a Reovirus, anOrthomyxovirus or a Paramyxovirus, such as a HIV1, HIV2, SIV, SHIV oranother virus described herein or in the cited references).

53C. The method of embodiment 52C wherein the subject has animmunosuppression condition that is associated with or caused by apathogen infection.

54C. The method of any of embodiments 46C-53C wherein the subject is amammal, a human, a primate or a rodent.

55C. The method of any of embodiments 46C-54C wherein about 0.05mg/kg/day to about 20 mg/kg/day is administered parenterally (e.g., byintravenous, subcutaneous, intramuscular, or intramedullary injection),topically, orally, sublingually or bucally to the subject, e.g., about0.1 mg/kg/day, about 0.2 mg/kg/day, about 0.5 mg/kg/day, about 1.0mg/kg/day, about 1.5 mg/kg/day, about 2 mg/kg/day, about 2.5 mg/kg/day,about 3.0 mg/kg/day, about 4 mg/kg/day or about 6 mg/kg/day, i.e., about0.1-10 mg/kg/day, typically about 0.2-7 mg/kg/day.

56C. The method of embodiment 55C wherein the subject is concurrentlytaking one or more second therapeutic agents to treat a pathogeninfection, e.g., a viral infection, such as a HIV-1 infection, a HIV-2infection, a HAV infection, a HBV infection, a HCV infection, an EpsteinBarr virus infection, a HSV-1 infection, a HSV-2 infection, humanherpesvirus 6 infection, human herpesvirus 7 infection, humanherpesvirus 8 infection, or a bacterial infection or a parasiteinfection, such as a malaria infection, leishmaniasis,cryptosporidiosis, toxoplasmosis, a mycoplasma infection, a Trichomonasinfection, a Chlamidya infection, a Pneumocystis infection, a Salmonellainfection, a Listeria infection, an Escherichia coli infection, aYersinia infection, a Vibrio infection, a Pseudomonas infection, aMycobacterium infection, a Haemophilus infection, a Neisseria infection,a Staphylococcus infection or a Streptococcus infection.

57C. The method of embodiment 56C wherein the one or more secondtherapeutic agents is a protease inhibitor, a reverse transcriptaseinhibitor, a viral, bacterial or parasite DNA or RNA polymeraseinhibitor, an antibacterial antibiotic or an antifungal agent, such asAZT, ddI, ddC, D4T, 3TC, a viral (e.g., HIV) fusion inhibitor,hydroxyurea, nelfinavir, saquinavir, ritonavir, indinavir, chioroquine,a chioroquine analog, amphotericin B, fluconazole, clotrimazole,isoniazid, dapsone, rifampin, cycloserine, erythromycin, a tetracyclineantibiotic, vancomycin, ethambutol, pyrazinamide, a flurorquinolone(e.g., ciprofloxacin, norfloxacin), a cephalosporin antibiotic, aβ-lactam antibiotic or an aminoglycoside antibiotic (e.g., streptomycin,kanamycin, tobramycin).

58C. The method of any of embodiments 46C-57C wherein the subject is ahuman, a primate, a canine, a feline or a rodent.

59C. A composition comprising an effective amount of an immune cellsubset modulatory compound of formula 1 and a pharmaceuticallyacceptable carrier.

60C. The composition of embodiment 59C wherein the immune cell subset is(1) CD8⁺ T cells, (2) CD4⁺ T cells, (3) CD8⁺ lymphokine activated killcells, (4) CD8⁻ lymphokine activated killer cells, (5) CD8⁻, CD16⁺natural killer cells, (6) CD8⁺, CD16⁺ natural killer cells, (7) CD8⁻,CD16⁺ cells that mediate antibody-dependent cell-mediate cytotoxicity,(8) CD8⁺, CD16⁺ cells that mediate antibody-dependent cell-mediatedcytotoxicity, (9) dendritic cells or dendritic cell precursors, (10)CD45RA⁺ T cells, (11) CD45RO⁺ T cells, (12) CD45RA⁺, CD45RO⁺ T cells,(13) CD8⁺, CD62L T cells, (11) CD4⁺, CD62L⁺ T cells or (14) HLA-DR⁺,CD8⁺, CD38⁺ T cells.

61C. A method to detect a biological response associated with theadministration of a compound of formula 1 to a subject comprising (1)obtaining a sample from the subject, (2) administering the compound offormula 1 to the subject to obtain a treated subject (3) obtaining asecond sample from the treated subject, (4) within 24 hours of obtainingthe sample, analyzing the sample to obtain control information fordetecting the biological response, (5) within 24 hours of obtaining thesecond sample, analyzing the second sample for the presence or absenceof a biological response to obtain experimental information and (6)optionally comparing the control information with the experimentalinformation to detect the presence, absence, relative magnitude orabsolute magnitude of the biological response

62C. The method of embodiment 61C wherein the compound of formula 1further comprises a pharmaceutically acceptable carrier.

63C. The method of embodiment 61C or 62C wherein the biological responseassociated with the administration of the compound of formula 1 to thesubject is modulation of the expression of a cell surface antigen, anincreased absolute or relative number of cells in an immune cell subset,a decreased absolute or relative number of cells in an immune cellsubset or an unchanged absolute or relative number of cells in an immunecell subset.

64C. The method of embodiment 63C wherein the immune cell subset is CD8⁺T cells, CD4⁺ T cells, CD8⁺ lymphokine activated killer cells, CD8⁻,CD16⁺ natural killer cells, circulating dendritic cell precursors,circulating dendritic cells, tissue dendritic cell precursors, tissuedendritic cells, CD8⁺ lymphokine activated killer cells, CD8⁻ lymphokineactivated killer cells, CD8⁻, CD16⁺ natural killer cells, CD8⁺, CD16⁺natural killer cells, CD8⁻, CD16⁺ cells that mediate antibody-dependentcell-mediated cytotoxicity, CD8⁺, CD16⁺ cells that mediateantibody-dependent cell-mediated cytotoxicity, CD45RA⁺ T cells, CD45RA⁺,CD45RO⁺ T cells, CD45RO⁺ T cells, CD8⁺, CD62L T cells, CD4⁺, CD62L⁺ Tcells or HLA-DR⁺, CD8⁺, CD38⁺ T cells, monocytes or macrophages.

65C. The method of embodiment 64C wherein the biological response is atleast transient modulation of an immune cell antigen or an immuneaccessory cell antigen (e.g., an adhesion molecule at the surface ofendothelial cells or a cytokine receptor at the surface of T cells or Bcells).

66C. The method of embodiment 65C wherein the immune cell antigen is aprotein, glycoprotein or cell surface antigen usually or only expressedby lymphoid cells (lymphocytes or white blood cells or their precursors,e.g., T cells, B cells, monocytes, macrophage, LAK cells, NK cells,dendritic cells).

67C. The method of embodiment 65C wherein the immune cell antigen is aCD molecule, an interleukin or a cytokine, optionally selected fromCD16, CD25, CD38, CD62L, CD69, CD45RA, CD45RO, IL-1, IL-2, IL-4, IL-6,IL-8, IL-10, TNFα, IGF₁ and γIFN.

68C. The method of any of embodiments 61C-67C wherein the subject is ahuman, a primate, a canine, a feline or a rodent.

69C. A method to alter the Th1-Th2 balance in a subject comprisingadministering an effective amount a compound of formula 1 to a subjectwhereby the subject's expression or secretion of IL-4 or IL-10 isdetectably modulated.

70C. The method of embodiment 30 wherein the subject's expression orsecretion of IL-4 or IL-10 is decreased and the Th1-Th2 balance in thesubject's Th1 immune responses to an infection or immunosuppressioncondition is detectably enhanced.

71C. The method of any of embodiments 1C-70C, wherein the formula 1compound is a compound named in any of compound groups 1 through42-25-10-6, or the formula 1 compound is a species in any genusdecsribed in any of compound groups 1 through 42-25-10-6.

Variations and modifications of these embodiments, the claims and theremaining portions of this disclosure will be apparent to the skilledartisan after a reading thereof. Such variations and modifications arewithin the scope and spirit of this invention. All citations herein areincorporated herein by reference in their entirety. All citations hereinare incorporated herein by reference with specificity.

EXAMPLES

The following examples further illustrate the invention and they are notintended to limit it in any way.

Example 1

BrEA formulation. Two lots of a non-aqueous BrEA formulation were madeat a BrEA concentration of 50 mg/mL in 25% polyethylene glycol 300,12.5% dehydrated ethyl alcohol, 5% benzyl benzoate, and 57.5% propyleneglycol as follows. BrEA was obtained from Procyte, Inc. The remainingexcipients are shown below.

Supplier Final Product Excipient Specification Lot No. ConcentrationPropylene glycol USP Arco Chemical 57.5% (v:v) HOC-61220-01104Polyethylene glycol NF Union Carbide   25% (v:v) 300 695752 Dehydratedalcohol USP McCormick 12.5% (v:v) Distilling 97K10 Benzyl benzoate USPSpectrum   5% (v:v) Pharmaceuticals MG025

The formulation was prepared by suspending BrEA in polyethylene glycol300, and sequentially adding propylene glycol, benzyl benzoate, anddehydrated ethyl alcohol to form a solution, which was diluted to thefinal desired volume with additional propylene glycol. The procedure isdescribed below.

The calculated amount of polyethylene glycol 300 was added to acompounding vessel. Then, while mixing, the calculated amount of BrEAwas added to the vessel, and mixed for at least 5 minutes to form asmooth, creamy liquid propylene glycol was added to the vessel, andmixed for a minimum of 5 minutes to form a uniform suspension. Thecalculated amount of benzyl benzoate is added to the vessel, and mixedfor approximately 5 minutes to form a translucent liquid suspension.Dehydrated alcohol was added to the vessel, and mixed for approximately5 minutes to form a clear, colorless solution. Propylene glycol was thenadded to achieve the desired final formulation, and mixed forapproximately 5 minutes. The drug solution was transferred to avolume-dispensing device set to deliver 1.2 mL per vial. Under nitrogenpressure, the solution was filtered through two 0.2 μm polyvinylidenefluoride filters in series into 2 cc amber glass vials. The vials werecapped with Teflon-coated, butyl-rubber stoppers and crimp sealed.Materials used in the product vials are listed below.

Material Source Product Code Description Vial Wheaton 2702-B51BA Tubingvial, 2 mL/13 mm, glass, type 1 amber Stopper Omniflex V9239 FM257/2 13mm, Teflon coated, butyl rubber stopper Seal West 4107 Flip seal, 13 mm,mist gray bridge

Product specifications were examined by one or more of the followingassays.

Test Specification Method Physical Examination Clear colorless solutionwith slight alcoholic odor Volume recovery NLT* 1.0 mL USP23<1> Specificgravity TBD USP23<841> Assay for active component 90-110% of label HPLCSterility sterile USP23<71> Endotoxin <0.1 EU/mg USP23<85> Particulatematter ≧10 μm NMT** 6000/cnt USP23<788> ≧25 μm NMT 600/cnt *NLT—no lessthan **NMT—no more than

Lot Analysis Test Specification Lot 1 Lot 2 Physical Clear colorlesssolution Positive Positive Examination with slight alcoholic odor Volumerecovery NLT 1.0 mL 1.15 mL — Specific gravity TBD  1.0411 — Assay foractive 90-110% of label 103.10% 104.25% component Sterility sterilesterile — Endotoxin <0.1 EU/mg 0.024 EU/mg — Particulate matter ≧10 μmNMT 6000/cnt  26 — ≧25 μm NMT 600/cnt  15

Example 2

BrEA drug substance and BrEA formulation stability. An acceleratedstability study of 6 months duration is conducted using BrEA and theformulations from example 1. Samples are taken at 1, 2, 3, 4, 5, and 6month time points and compared with the specifications listed inexample 1. Real time stability (25° C., 60% relative humidity) isconducted using BrEA formulation Lots 1 and 2, with sampling time pointsat 3, 6, 9, 12, 18, 24, and 36 months. After 3 months of storage at 40°C. and 75% relative humidity, the assay potency of BrEA is at least 95%of the label value. The results from the stability testing indicate thatBrEA is stable for at least 3 months at elevated temperature andhumidity in the Lot 1 and 2 formulations.

Example 3

Primate intermittent dosing protocol. Pig-tail Macaque monkeys infectedwith the SHIV₂₂₉ retrovirus were treated with a BrEA formulation asdescribed in example 1. SHIV₂₂₉ is a recombinant retrovirus containingHIV and SIV sequences. J. Thompson et al., abstract #75, 16^(th) AnnualSymposium on Nonhuman Primate Models for AIDS, Oct. 7-10, 1998, Atlanta,Ga., M. Agy et al., abstract #67, 16^(th) Annual Symposium on NonhumanPrimate Models for AIDS, Oct. 7-10, 1998, Atlanta, Ga. In monkeys, itestablishes an aggressive infection that leads to severe symptoms ofend-stage disease in infected untreated animals at about 180-210 daysafter infection. Four pig-tail macaques (2/group) received subcutaneousinjections of the formulation at 1 or 2 mg/kg body weight for 10consecutive days (Protocol 1). On week 8, 3 of the 4 monkeys wereretreated and 2 treatment naïve monkeys were treated with 5 mg/kg of theformulation on an every other day basis for a period of 20 days(Protocol 2). On week 19, all primates receiving treatment began a 3course treatment regimen with 3 mg/kg the BrEA formulation once dailyfor 10 consecutive days, repeated every four weeks for a total of 3treatment courses (Protocol 3).

The animals were infected with 1-100 TCID₅₀ units administeredintravenously or intrarectally. Viral titers in the first group ofanimals ranged from 10⁶ to 10⁸ before dosing began. All animalsdemonstrated an initial rise in plasma viral SHIV RNA. After a period of2 to 3 weeks, titers began to decline and 3 of the 4 animals showed aresponse to therapy with average viral titers of 0.76 log below baselineat weeks 4 to 5 after initiation of treatment. By week 8, titers in allanimals had returned to baseline values. Blood glucose levels droppedsignificantly, alkaline phosphatase levels were elevated and SGOT/GGTvalues trended towards the high end of normal. No other significantchanges were observed in any of the parameters monitored. The CD4 levelsin all monkeys remained less than 100 cells/mm³ at the end of the firstprotocol.

Three of the five monkeys on the second regimen (Protocol 2) respondedto the BrEA therapy with a greater depth and duration of response thanobserved at the lower dose levels. In the responding animals, theaverage decline below baseline was 1.47 log. The non-responding animalfrom Protocol 1 responded when administered the BrEA formulation inProtocol 2. Two animals did not respond, one each from the treatmentexperienced and treatment naïve groups. The third regimen (Protocol 3)is ongoing and animals are being monitored.

The monkeys on this study were salvaged from an infectivity study andthe first cohort of four monkeys on study (Protocol 1) were expected tolive only a few weeks past the initiation of these experiments as theywere beginning to deteriorate due to disease related causes. One animaldied at day 356 from a toxic reaction to the anesthetic used duringacquisition of a blood sample for analysis. At the time of thisapplication, the remaining monkeys are receiving multiple rounds oftherapy appear to be in good clinical health. Their survival was greaterthan 380 days from the time of infection. Treatment by intermittentdosing of the BrEA formulation was used. Three control monkeys wereinfected with 1-10,000 SHIV₂₂₉ TCID₅₀ units and did not receivetreatment. These animals are considered the no treatment arm of asurvival study. The mean time to death for pig-tailed macaques infectedwith SHIV₂₂₉ was 193 days. Monkeys receiving therapy remained in goodclinical health for over 350 days with CD4 levels less than 20 cells/mm³and without opportunistic infections or disease-related symptoms, otherthan a mild anemia in one animal.

These results show completely unexpected therapeutic responses by theprimates infected with the SHIV retrovirus, which is quite virulent. Theresults show that the majority of subjects in these treatment protocolsnot only had significantly prolonged survival compared to untreatedcontrols, but also the clinical symptoms associated with retroviralinfection improved dramatically, despite the fact that CD4 countsremained low, i.e., less than about 100 CD4 cells/mm³ initially and lessthan about 20 CD4 cells/mm³ later in the treatment protocols. To date,results such as this, i.e., (1) good clinical health in a majority ofsubjects having low CD4 levels (less than about 150 cells/mm³,especially less than about 75 cells/mm³) and (2) no clinical sign ofviral resistance to treatment despite intermittent dosing over aprolonged time period, are unprecedented in primates, humans or anyother animal. The SHIV₂₂₉ model is extremely pathogenic in pig-tailedmacaques. Events that occur in this model over several weeks wouldtypically take several years in humans infected with HIV. Treatment ofmonkeys infected with this virus and treated with commonly usedantiretrovirals, e.g., AZT, 3TC or a protease inhibitor, are notexpected to significantly affect the course of disease progression. Theclinical condition of the animals continues to improve, e.g., weightgain is about 8-15% per animal. These results show that the treatmentusing the intermittent dosing protocol is highly effective despite theapparent impairment of the subject's specific immunity, as shown by thelow CD4 counts. Increased CD4 counts may be attained using immunestimulators such as IL2 or they may increase spontaneously in somesubjects such as humans, depending on the treatment protocol, theduration of dosing or the subject's initial medical condition. Theantiviral effects shown here appear to function at least in part byenhancing the subject's immune responses, e.g., enhanced immune responseby phagocytic cells (NK cells, monocytes and/or macrophages), and/orenhancing any residual specific immune responses, if any, that thesubject may be able to muster.

Example 4

Human treatment protocol. A dose escalation clinical trial is performedusing a nonaqueous formulation containing BrEA or another formula 1compound(s) that is prepared essentially as described in example 1. Thepatients are treatment naive or treatment experienced and about 3-10patients are examined at each dose level. The initial dose is 25 mg ofBrEA or another formula 1 compound(s) that is administered parenterally,e.g., s.c. or i.m. The dose is administered once or once or twice perday for 1-12 days, followed by no dosing for at least 7 days (e.g., 7 to90 days). Subsequent doses are administered once or once per day for1-12 days, followed by no dosing for at least 7 days (e.g., 7 to 90days). Other dose levels tested are 20 mg, 50 mg, 75 mg, 100 mg, 150 mg,200 mg, 250 mg and 300 mg with each dose administered once per day as asingle dose or as two, three or more subdivided doses. An efficacydosing trial is performed using the same dosing protocol as the doseescalation trial or it may alternatively comprise dosing once or twiceevery other day for 3-17 days, followed by no dosing for 7-90 days andthen repeating the dosing once or twice every other day for 3-17 days.This protocol is repeated indefinitely (e.g., at least about 3-18months) using the optimal dose(s) obtained from the dose escalationtrial, e.g., about 10-200 mg/day of a formula 1 compound.

Example 5

Animal pharmacological studies. Nonclinical studies were conducted usingan oral and a subcutaneous formulation of BrEA. Rats were orallyadministered ¹⁴C BrEA solubilized in different excipients to determinethe levels of drug in blood and various tissues. The results of thesepreliminary pharmacokinetics studies indicated that the absorption ofBrEA by oral administration is about 0.1 to 15%, with at least about 80%excreted in the feces.

The nonaqueous BrEA formulation of example 1 was administered as asingle subcutaneous dose to rabbits. More than 90% of the drug left theinjection site within 24 hours of administration, and reached a maximumconcentration in the plasma of about 1.2% of the injected dose at eightto twelve hours post administration. The circulating half-life of thedrug in the plasma was about twelve hours. The drug did not accumulateto a significant extent in any major organ and was primarily excreted inthe urine.

BrEA was administered subcutaneously to rats using the formulation ofexample 1. Approximately 90% of the drug left the injection site within24 hours of administration, and reached a maximum concentration in theplasma of about 0.2% of the injected dose at one hour postadministration. Elimination from the plasma was biphasic, withhalf-lives of about 12 and 72 hours respectively. BrEA did notaccumulate to a significant extent in any major organ, and was excretedprimarily in the feces. A study is also performed in Rhesus Monkeys withthe example 1 formulation to determine plasma pharmacokinetics.

A pharmacokinetic analysis of ¹⁴C BrEA in plasma was conducted in twofemale Rhesus Monkeys. Trace labeled compound(16α-bromo-3-beta-hydroxy-5α-[4-¹⁴C]-androstan-17-one [50 mCi/mmole])was used at a dose of 1 mg/kg as a subcutaneous injection in thescapular region using an injection volume of 1 mL/kg. The BrEA wasformulated in 25% polyethylene glycol 300, 12.5% absolute ethanol, 5%benzyl benzoate, and qs with propylene glycol. 40 μCi were injected peranimal. Blood samples were taken at 0, 0.5, 1, 2, 4, 8, and 24 hours fordetermination of ¹⁴C activity. The radioactivity in the plasma rose tonear peak concentration in 8 hours and remained at approximately thesame level through the end of the study at 24 hours.

A pharmacokinetic analysis of ¹⁴C BrEA was conducted in New ZealandWhite rabbits. Twenty μCi of ¹⁴C16α-bromo-3-betα-hydroxy-5α-[4-¹⁴C]-androstan-17-one (50 mCi/mmole) plus1 mg/kg unlabeled BrEA was administered to each of three New ZealandWhite rabbits as a subcutaneous injection in the scapular region usingan injection volume of 1 mL/kg. The drug was formulated in 25%polyethylene glycol 300, 12.5% absolute ethanol, 5% benzyl benzoate, andqs with propylene glycol. Blood samples were taken at 0.5, 1, 2, 4, 8,12, 24 hours for all three animals, and at 48 hours for two of theanimals. Twenty-four and 48 hours after administration, one and twoanimals respectively, were sacrificed, and the following organs/tissueswere collected: brain, heart, kidneys, liver, lungs, skeletal muscle,spleen, and injection site muscle and skin. In addition to the organsand tissues, urine and feces were collected as well as the cage wash.BrEA did not accumulate to a significant degree in any of the organslisted above. Of the organs, the greatest mass of drug was observed inthe liver, containing approximately 0.8% and 0.12% of the injected doseat 24 and 48 hours, respectively (average 0.13%).

Percentage of Drug in Organs (Rabbits) Animal 201 Animal 301 Animal 302Organ or Tissue 24 hours 48 hours 48 hours Brain 0.005 0.002 0 Heart0.008 0.003 0.002 Kidneys 0.155 0.055 0.050 Liver 0.76 0.145 0.125 Lungs0.029 0.019 0.011 Spleen 0.002 0 0 Skeletal muscle 0.002 0 0 (sample wt.in grams) (3.8 g) (6 g) (5 g) Skin 0.008 0.002 0.004 (sample wt. ingrams)   (8 g) (6 g) (9 g)

The average percentages of the administered dose in whole blood wascalculated by multiplying the concentration of drug in whole blood bythe assumed volume of blood in the animals, 200 mL. The amount of drugin the blood reaches a maximum at around 8 hours, and a small amount wasstill evident at 48 hours. The amount of BrEA in whole blood wasconsistently lower than in plasma, suggesting the drug is not taken upto an appreciable extent by red blood cells.

In vivo experiments were conducted to determine the bioavailability ofBrEA via oral administration using different formulations. BrEA was (1)solubilized in soya oil, vitamin E oil, a mixture of vitamin E andcremophore or (2) BrEA was micronized and combined with or without asurfactant. These formulations are described below. The formulationswere administered orally to rats and BrEA levels were determined in theblood, liver, spleen, kidney, and the lymph nodes. In the studies usingmicronized BrEA, the brain was evaluated for drug uptake. Twenty-fourhour urine and feces were collected when BrEA was solubilized in vitaminE and soya oils and vitamin E mixed with Cremophore. The data from thesestudies indicate that BrEA enters into the lymphatics but is eliminatedrapidly from the other tissues. The amount of ¹⁴C radioactivityrecovered in the feces 24 hours after administration was 78 to 83%. Abrief summary of each experiment is provided below and the results areprovided in Table 6.

BrEA (5 mg in 1.0 mL of soya oil or vitamin E oil) supplemented with¹⁴C-labelled BrEA was administered intragastrically to rats.Solubilization of BrEA in the vitamin E or soya oil was facilitated with50 μL ethanol. Animals (3/time point) were assayed at 1.5, 3, 5.5, and24 hours after administration and the ¹⁴C-radioactivity was measured inthe blood, liver, spleen, kidney, lymph nodes and 24-hour feces andurine. The results indicate that, on the basis of ¹⁴C-radioactivity,some of the BrEA is taken into the lymphatic system. The uptake isgreater with soya oil than vitamin E oil in the blood, liver, and lymphnodes.

BrEA (5 mg in 1.0 mL of a vitamin E and cremophore) supplemented with¹⁴C-labelled BrEA was administered intragastrically to rats.Solubilization of BrEA in the vitamin E-cremophore mixture wasfacilitated by the adding 60 μL ethanol. Animals (4/time point) weresacrificed at 2, 3, 5.5, and 24 hours and ¹⁴C-radioactivity was measuredin the blood, liver, spleen, kidney, lymph nodes and 24 hour feces andurine. The results indicate that a small portion of the drug is taken upby the lymphatic system. Judging from the values in plasma, liver andlymph nodes, it appears that drug uptake is slower compared with soy oilor vitamin E and its presence in the tissues is more persistent.

Rats, in groups of three males, were orally administered 1.0 mL of 0.9%NaCl containing 10 or 32 mg BrEA micronized with a surfactant,Synperonic PE/F 127 (2.5% wt/wt). Rats were examined at 1.5, 5 and 24hours after administration. Blood, liver, spleen, kidney, lymph nodes,and brain were assayed for ¹⁴C radioactivity. The levels of BrEA in theblood, in comparison to the experiments with BrEA in Vitamin E oil andsoya, were higher, 0.3% at 1.5 hours, and increased after 5 hours to0.8% and 0.9% of the 10 and 32 mg dose, respectively. Additionally, thevalues in the lymph nodes were similar to those measured at 1.5 hoursand the levels were sustained at 5 hours (5.3 and 5.0%) and 24 hours(3.7 and 3.1%) for the 10 and 32 mg dose, respectively (refer to Table6).

In a repeated dose experiment, rats were intragastrically administered1.0 mL 0.9% NaCl containing 2 mg BrEA micronized with Synperonic PE/F127 (2.5% wt/wt) every 6 to 16 hours. Rats (3/time point) weresacrificed at 40, 72, 84, 90 and 96 hours after the firstadministration. Blood, liver, spleen, kidney and lymph nodes wereassayed for ¹⁴C radioactivity. Higher levels in the blood, liver,kidneys and lymph nodes were noted in this experiment over previousstudies.

Rats, in groups of three males, were orally administered 1.0 mL of 0.9%NaCl containing 2, 4 or 10 mg BrEA micronized without a surfactant. Ratswere sacrificed at 1.5, 5 and 24 hours after administration and blood,liver, spleen, kidney, lymph nodes and brain were assayed for ¹⁴Cradioactivity. The concentration of BrEA micronized without a surfactantin the observed tissues was lower than BrEA plus a surfactant.

Example 6

Inhibition of parasites in vitro. For in vitro antimalarial testing,micro-titer plates were used. The concentration of drugs was prepared aspMol/well according to WHO standard procedures (WHO, 1990). The testcompound was dissolved in 15% DMSO in sterile RPMI-1640. Bothchloroquine sensitive (e.g., WS/97) and resistant (e.g., MN/97) isolatesof Plasmodium species are used.

A schizont inhibition assay was performed as follows. The micro-titerplates were predosed with various concentrations of the test compound.50 μL of parasitised erythrocyte suspension in RPMI-1640 (0.2 mLerythrocyte+0.3 mL serum+4-5 ml RPMI-1640) were dispensed in microtiterwells that contained various concentrations of drug. Triplicate readingswere made for each concentration.

A ³H-hypoxanthine incorporation assay was performed as follows. Thetesting was carried out according to the procedure of Desjardins et al.1979. After 30 hr culture at 37 degrees C., the same microtiter platesfrom schizont inhibition assays with another triplicate wells werepulsed with ³H-hypoxanthine for overnight. The cell suspensions werewashed twice on millipore glass fiber filter with Millipore filterapparatus. The filter discs were counted for DPM by a Beckman LS6000β-scintillation counter. The activity of the drug was measured byplotting DPM against concentration of drug.

Activity of compounds against Chloroquine sensitive T996/86 P.falciparum in vitro Etianic Acid Concentration Etienic Acid Methyl (μM)DHEA* BrEA* Methyl Ester* Ester* 30 65.6 98 60 61.5 15 44 60.1 45.7 47.47.5 38.3 50 40.9 45.3 3.25 37.2 43.7 46 41.4 1.875 23.2 40.9 41 43.40.938 37.2 31.8 43.3 47.1 IC₅₀ 19.0 μM 7.5 μM 19.5 μM 17.5 μM *%inhibition Concentration (nM) % Inhibition Chloroquine Chloroquine 20095.9 100 94.6  50 97.3  25 94.5  12.5 86.8  6.25 27.2 IC₅₀ 9.0 nM

The activity of 16α-chloroepiandrosterone and16α-bromodehydro-epiandrosterone against chloroquine sensitive T996.86and chloroquine resistant KI P. falciparum in vitro is shown below.

T996.86 KI 16-chloroepiandrosterone IC₅₀ −9.25 pg/mL ~9.25 μg/mL DHEA-BrIC₅₀ −25.0 pg/mL ~25.0 μg/mL

Other formula 1 compounds, e.g., any compound in compound group 1through 25-6 are used in a similar manner to inhibit Plasmodiumparasites.

Example 7

Four-day in vivo protocol for inhibition of Plasmodium berghei. The4-day suppressive test has been widely used and it can be performedwithin a 1-week period. The test consists of the inoculation ofparasitised erythrocytes on the first day of the experiment (D₀),followed by an injection of the test compound, which is alsoadministered on the 2^(nd), 3^(rd) and 4^(th) days of the protocol. Onthe 5^(th) day, blood films are taken and antimalarial activity isassessed either by calculating parasitemia, or by scoring parasitenumbers on a predetermined scale (i.e., 1-5). Peters (Ann. Trop. Med.Parasitol. 64: 25-40,1970) described a basic procedure using this 4-daytest.

The protocol is summarized as follows. Five female TO mice were used pertest group. P. berghei HP15 ANKA parasites were collected by cardiacpuncture using a heparinised syringe from a donor mouse having a 30+%parasitaemia. The blood was diluted with diluting agent (50% HIFCS+50%sterile PBS) to a final concentration of 1% parasitaemia or 1×10⁷infected erythrocytes per 0.2 mL of the infecting suspension. Each mousewas inoculated intravenously, which produced a more uniform infectionrate than intraperitoneal administration of 0.2 mL of the infectingsuspension. Test compounds were prepared at doses of 100 mg/kg in (16.7%DMSO+83.3% Celacol). The steroid formulations were administeredintraperitoneally 2 hours after parasite inoculation. The compounds wereadministered once a day starting on D₀, and continued on the followingthree days. Blood films were made from tail blood on the day after thelast dosing of compound and the blood was fixed with 100% methanol andstained with 10% Giemsa. Parasitaemias were scored on a scale of 0-5,where 5 is equal to the control.

An inoculum of 1% parasitaemia 1×10⁷ erythrocytes/mL, 0.2 mL per mouse(female strain TO mice), was delivered by intravenous injection. Drugadministration commenced 2 hours after inoculation on Day 1 andcontinued for 3 days. The results are shown below from blood films fromall 20 mice on Day 5 when parasitaemias were assessed.

Compound Treatment Parasitaemia Score (0-5) BrEA 100 mg/kg × 4 daysi.p.* 1 Etienic Acid 100 mg/kg × 4 days i.p. 2 DHEA 100 mg/kg × 4 daysi.p. 1 Chloroquine  3 mg/kg × 4 days i.p. 1 control N/A 5 *i.p. =intraperitoneal injection

In a similar protocol, mice are inoculated with a solution containing1×10⁷ erythrocytes/mL by I.V. injection. Two hours later give drug isdelivered by I.V. injection. BrEA or another formula 1 compound is given(0.2 mL I.V. or S.C.) once a day for 4 days. Tail snips are used toobtain blood after the study. Mice infected with P. berghei were used toobtain infected cells. Parasites are harvested from cardiac mouse blood,and uninfected mice are infected using 0.2 ml of blood with 14%parasitaemia per mouse I.V. Two hours later, the first dose of BrEA (100mg/kg I.V. or S.C.) is delivered to the infected animals. The BrEAformulation was a sterile solution containing 15 mg/mL of BrEA in 45%hydroxypropyl-β-cyclodextrin and 0.9% saline. At 1, 2, 3 and 4 daysafter the infection of the animals, BrEA (100 mg/kg I.V. or S.C.) isdelivered to the infected animals. No deaths occurred in the groupreceiving I.V. BrEA at day 30, but all control animals were dead by day10. All animals treated with BrEA by S.C. delivery were dead by Day 11.

Example 8

Rat in vitro and in vivo study. In the in vitro protocol the parasite(Plasmodium falciparum, chloroquine sensitive strain WT and chloroquineresistant strain Dd2) level is adjusted to 1% and the hemocrit isadjusted to 7% with medium. Using a 96 well plate, 50 μL of parasite and100 μL of drug mixed with media are added to each well and the procedureis done in triplicate. The plate is placed in a chamber containing aphysiological gas mixture and incubated at 37° C. The media/drug mixtureis changed at 24, 48 and 72 hours. On day 5 (96 hours) slides of eachwell are made, stained with Gemsia and 500 red blood cells are countedfor each slide. The triplicates are averaged and data are reported inpercent inhibition.

In the in vivo protocol, Lewis rats weighing 80-85 grams were given astandardized IP injection of parasite (Plasmodium berghei). Rats werethen intravenously injected 2 hours later with one of the treatmentsdescribed in the table below, returned to their housing, fed standardlab chow and allowed free access to water. Animals were weighed andtreated again 24, 48, and 72 hours after the first treatment and againreturned to their housing and they were allowed free access to food andwater. The animals were weighed again and then bled using a 26-gaugeneedle on day 5,11 and 28 post inoculation. Hemocrits were measured andblood smears are prepared for each rat. The blood smears were thenstained using Gemsia and the level of parasitemia (defined as thepercent of red cells with parasites) was determined. Animals were againreturned to their housing and observed twice daily for evidence ofprogressive disease, defined as listlessness and or adverse drugreaction, which is defined as a loss of 20% of original body weight, fora total of 28 days. If either progressive disease or drug reaction isnoted, the animals are euthanized.

The BrEA formulation was a sterile solution containing 15 mg/mL of BrEAin 45% hydroxypropyl-β-cyclodextrin and 0.9% saline.

Group 1 Group 2 Group 3 Group 4 Control 0.9% Chloroquine BrEA Low BrEAHigh saline Control Dose Dose 40 mg/kg 30 mg/kg 60 mg/kg

The intravenous injections were given on days 0, 1, 2 and 3 and theresults are shown below. The results showed that treatment in vivo witha formulation comprising BrEA reduced parasitemia to a level comparableto that seen with the chloroquine (“Clq”) control. The results aresummarized below.

% RBC parasitemia Day 4 saline control 16% chloroquine control 10% lowdose BrEA 9% high dose BrEA 7% Day 11 saline control 36% chloroquinecontrol 16% low dose BrEA 12% high dose BrEA 11%

Example 9

Human clinical study—parasite infection. Response to drug treatment wasgraded as per World Health Organization criteria (WHO 1973) in infectedpatients. Evaluation of therapeutic response was determined using theparasitic and fever clearance times. Parasite clearance was expressed asthree indices; the time for the parasite count to fall by 50% of thepre-treatment (baseline) value (PC₅₀), (ii) the time for the parasitecount to fall by 90% of the baseline value (PC₉₀) and (iii) the time forthe parasite count to fall below the level of microscopic detection(parasite clearance time PCT) (N. J. White and S. Krishna Trans. R. Soc.Trop. Med. Hyg. 83: 767-777, 1989; White et al., J. Infect. Dis. 165:599-600, 1992; White et al., J. Infect. Dis. 166: 1195-1196, 1992). Thefever clearance time was defined as the time from drug administrationtill the oral or rectal temperature fell to or below 37.2° C. andremained so for at least 48 h.

Venous blood (5 mL) was obtained from two patients before treatment andat 4, 6, 8, 12, 18, 20, 24, 30 and 36 h after treatment or at 4 or6-hourly intervals after treatment until there was complete clearance ofperipheral parasitemia. Blood was collected aseptically and transferredto 10 mL syringes containing 2 mL of acid citrate dextrose (ACD) for invitro culture. Prior to incubation, the plasma was separated from thered blood cells and the red blood cells were washed twice. Parasiteswere cultured by modification of standard in vitro culture techniques(W. Trager and J. B. Jensen, Science 193:673-675, 1976; A. M. Oduola etal., J. Protozool. 39: 605-608, 1992). Samples were dispensed intosterile centrifuge cubes within 10 min of collection and spun down. Thesupernatant plasma was stored while the packed cells were washed twicewith culture medium (washing medium, RPMI-1640 medium, containing 25 mMHEPES buffer and 25 mmol/L NaOH). The buffy coat was removed by vacuumaspiration. A 1:10 fold dilution was done for each blood sample withcomplete washing medium [CMP (washing medium supplemented with 10% humanplasma)]. One milliliter each of the sample was transferred into 2 wellsof a 24 well micro culture plate. Cultures were incubated at 37 degreesC. in an atmosphere of 5% CO₂, 5% O₂ and 90% N₂ premixed gas. Theculture medium was changed daily and thin blood smears were prepared formicroscopy at 24 and 48 h after the culture has been set up. The culturesamples were diluted with unparasitized washed type A Rh-positive redblood cells if the proportion of parasitized red blood cells was morethan 2%.

Microscopy. During the in vivo study, thin and thick blood films werefixed with dehydrated methanol (100%) and heat, respectively, werestained with 10% Giemsa for 20 min. Parasitemia was quantified in thinfilms by counting 2000 red blood cells in clear contiguous fields andfinding the proportion that was parasitized. In thick films, parasitemiawas quantified by counting parasites against leukocytes. A film wasdeclared negative if no parasites were found after examination of 200microscope fields of a thick smear. During in vitro and ex vivo study,pretreatment thin and thick smears were, graded for ring stages by themethod of Jiang as modified by Li et al. (J. B. Jiang et al, Lancet2(8293): 285-288, 1982; K. Silamut and N. J. White Trans. R. Soc.Trop.Med. Hyg. 87: 436-443, 1993; X. L. Li et al, Chi. J. Parasitol.Dis. 12: 296, 1994). Approximately 5000 erythrocytes were counted inclear contiguous fields 24 and 48 h after incubation of blood obtainedat each time point and graded for maturity into tiny rings, small rings,large rings, pigmented trophozoites and schizonts. Functional viabilitywas estimated as the percentage of asexual ring forms capable ofmaturing to pigmented trophozoites or schizonts after 24-48 h of invitro culture (W. M. Watkins et al., Trans. R. Soc. Trop. Med. Hyg. 87:75-78, 1993).

Calculation of parameters. The patients presented with acute symptomaticsevere non-cerebral pure P. falciparum malaria. They had oral fluidintolerance, body temperatures greater than 39° C., greater than 5000parasites per micro liter of blood, asexual parasitemia and they had anegative urine test for antimalarial drugs. They were administered 25 mLintravenously every four hours with BrEA suspended in sterile 45%β-cyclodextrin in saline at a concentration of 25 mg/mL. This regimenwas continued for four days. Parasitemia quantification and clinicalexamination were done once every 6 hours for the first 72 hours,followed by daily assessment of the parameters up to day 7 (168 hrs) andthereafter on day 14.

Blood films were Giemsa-stained and parasitemia quantification was donein thick films by counting 2000 parasites against leukocytes, and thethin films by finding the proportion of infected red blood cells.Response to drug treatment was graded according to WHO criteria.Evaluation of therapeutic response was done using the parasitic andfever clearance times. Parasite clearance was expressed as threeindices: The time for the parasite count to fall by 50% of thepre-treatment (baseline) value (PC₆₀); to fall by 90% of the baselinevalue (PC₉₀); and to fall below the level of microscopic detection(parasite clearance time) PCT.

The fever clearance time was defined as the time from drugadministration until the oral/rectal temperature fell to below 37.2degrees C. and remained so for greater than 48 hours. The parasiteclearance rate at day 14 was 100%. The clinical response thus includedan effect on parasitemia in both patients and amelioration of one ormore symptoms of infection.

Intravenous BrEA Malaria Patient Trial Patient A Patient B Feverclearance time 12 hrs 18 hrs Parasite clearance times Time to 50%clearance 18 hrs 24 hrs Time to 90% clearance 24 hrs 48 hrs Time to 100%clearance 48 hrs 64 hrs

Example 10

Cellular studies in vitro. The effect of BrEA on pentosephosphate shunt(PPS) activity in normal human RBC was examined using whole cells. Sinceglucose-6-phosphate dehydrogenase (“G6PD”) is the limiting enzyme of thePPS, PPS flux measurement is considered to better reflect G6PD activityin the whole cell compared to G6PD activity measurement in a celllysate. G6PD activity measured in a cell lysate is typically about1100-fold higher than the PPS flux in whole resting unstimulated RBC(G6PD activity in cell lysate: 165; PPS flux 0.142 micromoles/hour/mlRBC). PPS flux and G6PD activity in the whole RBC depends on a number offactors (the concentration of NADPH, NAD, and ATP, and intracellularpH), which are kept constant if the measurement is performed in thelysate and may vary in the whole RBC. Levels of G6PD activity in cellsis considerably above normal basal needs and inhibition of overall G6PDactivity might have no or minor consequence on PPS flux in the wholecell. For example, RBC with the Mediterranean G6PD mutant with about 1-3percent residual activity compared with normal individuals have noimpairment in basal PPS flux, but show impaired flux when flux throughPPS is stimulated by methylene blue addition. A series of experimentswere perfromed using varying amounts of BrEA and PPS flux was measuredin unstimulated basal RBC and in methylene-blue (MB)-stimulated RBC.

The data below shows PPS flux (micromoles/hour/ml RBC) in basalunstimulated, and MB-stimulated normal RBC. Different concentrations ofBrEA (0.3, 3.5 and 7 micromolar, final) were supplemented to suspensionsof washed RBC suspended in RPMI, pH 7.4 at 10% hematocrit, whereby PPSflux was immediately measured without further incubation and withoutfurther washings. A minor inhibition of MB-stimulated PPS flux wasobserved with BrEA at 7 μM.

PPS flux control, unstimulated RBC 230 DMSO control, unstimulated RBC270 DMSO control, MB stimulated RBC 5090 0.3 μM BrEA, unstimulated 2500.3 μM BrEA, MB stimulated 5000 3.5 μM BrEA, unstimulated 270 3.5 μMBrEA, MB stimulated 4950   7 μM BrEA, unstimulated 295   7 μM BrEA, MBstimulated 4660

The data below shows average values of 3 experiments, where basal,unstimulated, and MB-stimulated PPS flux (micromoles/hour/ml RBC) wasmeasured in normal RBC. In these experiments, different concentrationsof BrEA ˜0.8, 8 and 80 micromolar, final) were supplemented tosuspensions of washed RBC suspended in RPMI, pH 7.4 at 10% hematocrit.After a 90-min incubation at 37° C. with and without BrEA, PPS flux wasmeasured. The results showed a dose-dependent inhibition ofMB-stimulated PPS flux. Inhibition was 10% at 8 micromolar (p=0.006 vscontrol+DMSO) and 25% at 80 micromolar (p=0.002 vs control+DMSO).

PPS flux control, unstimulated RBC 430 control, MB stimulated RBC 5410DMSO control, unstimulated RBC 480 DMSO control, MB stimulated RBC 48900.8 μM BrEA, unstimulated 410 0.8 μM BrEA, MB stimulated 4930   8 μMBrEA, unstimulated 450   8 μM BrEA, MB stimulated 4430  80 μM BrEA,unstimulated 450  80 μM BrEA, MB stimulated 3660

Example 11

Inhibition of parasite growth. The effect of Epi(16α-bromo-epiandrosterone) on parasite (Plasmodium falciparum) growthwas shown. EPI was active at a concentration of 1 μM.

Parasitemia after treatment Time 0 24 hrs 48 hrs 72 hrs control + DMSO5% 5.40% 3.10% 5.20% Epi 1 μM 5% 5.70% 5.50% 1.60% Epi 10 μM 5% 5.60%0.90% 0 Epi 100 μM 5% 0 0 0 Epi 500 μM 5% 0 0 0 control + DMSO 2% 8.80%  11%   8% Epi 50 nM 2% 9.90% 9.20% 8.30% Epi 1 μM 2% 5.80% 6.10% 2.10%Epi 2.5 μM 2% 7.30% 5.80% 3.20% Epi 5 μM 2% 5.40%   6% 1.80% Epi 10 μM2% 4.20%   3% 0 Epi 50 μM 2% 0 0 0

Parasitemias were determined by standard methods (microscopic inspectionof at least 500 cells, stained with DIFF-QUICK™ (Baxter). Parasites werecultured under standard conditions in RPMI-1640 supplemented withHepes/Glucose (10 mM), glutamine (0.3 g/liter) and 10% human plasma. Thehematocrit was 1%.

Example 12

Stimulation of phagocytosis. The capacity of BrEA to influencephagocytosis of Plasmodium parasite-infected RBC is examined usingadherent human monocytes. The parasitemia level is about 8-10% and humanmonocytes are obtained from buffy coats from blood as follows.Peripheral blood mononuclear cells are separated from freshly collectedplatelet-poor buffy coats discarded from blood samples of heafthy adultdonors of both sexes. Separated cells are washed once with luke-warm PBSsupplemented with 10 mM glucose (PBS-G) and resuspended at 5×10⁶cells/mL in ice-cold RPMI 1640 medium supplemented with 23 mM NaHCO₃ and25 mM Hepes, pH 7.4 (RMBH). Dynabeads M450 Pan B and Pan T (Dynal) areadded to cells in a 4:1 ratio for 20 min at 4° C. B-lymphocytes andT-lymphocytes are removed as specified by the manufacturer. Theremaining monocytes are washed 2 times in RMBH, resuspended in AIM Vcell culture medium (Gibco) at 1×10⁶ cell/mL. The monocyte layer iscollected, washed with PBS-G at 37° C. and resuspended in AIM V mediumat 1×10⁶ cells/mL. Purified cells are>90% monocytes as assessed by CD14expression.

Phagocytosis of opsonized parasitized RBC (PE) is determined as follows.Phagocytosis of fresh-serum opsonized PE is initiated by mixing 10PE/monocyte. Suspensions are briefly centrifuged (150×g for 5 sec atroom temperature) to improve contact between PE and monocytes. To avoidattachement of monocytes after centrifugation and during the wholeincubation period, cells are kept in suspension at 5×10⁶ cells/5 mL AIMV medium in 6 cm diameter teflon bottom dishes (Heraeus) in a humidifiedincubator (95% air, 5% CO₂) at 37° C. On average, at least 90% of themonocytes phagocytose PE, as assessed by microscopic inspection. Controlcells are kept under similar conditions without phagocytosis.Quantitative assessment of phagocytosis is performed by a previouslydescribed bioluminescence method (E. Schwarzer, et al., Br. J. Haematol.1994 88: 740-745).

Erythrocyte treatments and parasite cultures are as follows. Fresh blood(Rh+) is used to isolate erythrocytes (RBC). Washed RBC are infectedwith schizont/trophozoite parasite stages (Palo Alto strain,mycoplasma-free). Stage specific parasites are isolated by thePercoll-mannitol method. Briefly, normal schizont-stage parasitized RBC(SPE) separated on Percoll-mannitol gradient (parasitemia>95% SPE) aremixed with RBC suspended in growth medium (RPMI 1640 medium containing25 mmol/L Hepes, 20 mmol/L glucose, 2 mmol/L glutamine, 24 mmol/LNaHCO₃, 32 mg/L gentamicin and 10% AB or A human serum, pH 7.30) tostart synchronous cultures at selected hematocrit values. The inoculumparasitemia is adjusted to 20% normal SPE for isolation of ringparasitized RBC (RPE) and to 5% normal SPE for isolation oftrophozoite-stage parasitized RBC (TPE). At 14-18 hours after inoculumparasites are at ring-stage in the first cycle; at 34-33 hours,parasites are at trophozoite-stage in the first cycle; and at 40-44hours after inoculum parasites are at schizont-stage in the first cycle.RPE, TPE and SPE are separated on Percoll-mannitol gradients. Theparasitemia is usually 8-10% RPE, and >95% TPE. Nonparasitized andparasitized RBC are counted electronically. To assess total parasitemiaand relative contribution of RPE, TPE and SPE, slides are prepared fromcultures at indicated times, stained with DIFF-QUICK™ parasite stain andabout 400-1000 cells are examined microscopically.

The effect of a formula 1 compound such as BrEA in parasitized RBC isexamined using various concentrations of the compound, e.g., BrEA, e.g.,0.5 μM, 1 μM, 10 μM, 25 μM and 50 μM. Trophozoite-parasitized RBC,schizont-parasitized RBC or ring-parasitized RBC are examined asdescribed.

Example 13

Human malaria clinical trial. The clinical trial protocol thatincorporates about 15-20 patients is established. For a phase I, I/II orII trial, the patients are mildly infected with one or more Plasmodiumparasites and they are mildly symptomatic (less than about 8-10%parasitemia of RBC). Before treatment, the patients are optionallytested for infection with HIV, HCV, TB, and Cryptosporidium. Patientswith one or more co-infections are given standard care for thecoinfection. The patients are hospitalized for treatment for one week.Two or more dose groups, e.g., 25, 50 or 100 mg/day of BrEA administeredparenterally, e.g., by intramuscular, subcutaneous or intravenousinjection, on 3, 4 or 5 days of the week when patients are dosed. Dosingis on consecutive days or on an intermittent schedule, e.g., 2, 3 or 4doses with one dose administrered every other day.

The formulation containing BrEA is as described herein, e.g., theformulation of example 1 or a formulation that comprises 100 mg/mL BrEA,PEG300 ˜30% v/v, propylene glycol 30% v/v, benzyl benzoate 30% v/v andbenzyl alcohol 2% v/v. At day 5-7, if less than about 50% reduction inparasitemia is observed, the patients are given standard care formalaria (mefloquine). During the week of treatment and for 1, 2 3, ormore weeks there after, blood samples are taken periodically forevaluation of parasitemia, pharmacokinetics, plasma cytokines (e.g.,IL-2, IL-4, IL-10, IGF1, γIFN, GM-CSF), and intracellular cytokines(e.g., IL-2, IL-4, IL-10, IGF1, γIFN, GM-CSF). The patients areoptionally treated again at about 2 to 12 weeks after the initialdosing, using the same or a similar protocol as that used in the initialdosing protocol.

An exemplary open-label study of a BrEA formulation administeredintramuscularly to semi-immune patients with uncomplicated malaria isconducted. The formulation comprises 100 mg/mL BrEA, PEG300 ˜30% v/v,propylene glycol 30% v/v, benzyl benzoate 30% v/v and benzyl alcohol 2%.Patients will remain at the hospital as in-patients for the first 7 daysof the study. Patients will receive one daily intramuscularadministration of 50 mg or 100 mg of BrEA for 5 consecutive days. Dailyevaluation for the first 7 days, and up to study day 14, may includeparasitemia evaluation (twice daily), chemistry, hematology and druglevels (pharmacokinetic evaluation). If, after study day 7, theparasitemia levels decrease from the screening value and the patient isclinically stable, the patient may be followed on a daily basis forparasitemia (twice daily) for up to an additional 7 days as hospitalin-patients. If a patient becomes clinically unstable at any time duringthe study, the patient will be discontinued and may be offered thestandard treatment for malaria. Patients deficient inglucose-6-phosphate dehydrogenase enzyme may be excluded, since BrEAinhibits the enzyme. Other considerations that may lead to exclusion ofpatients from the trial include patients diagnosed with any of thefollowing: severe anemia (hematocrit<21% or hemoglobin<7 g/dL); renal orliver failure by history and/or laboratory results respiratory distressas evidenced by dyspnea or respiratory rate≧30 per minute; hypotension(systolic blood pressure<90 mm Hg); tachycardia (heart rate>130beats/minute); pregnant or breast-feeding women; significant activeco-morbid illness (acute medical diagnosis requiring specific therapy;patients with parasitemia>10% on peripheral smear.

Blood samples may be collected from each patient for future clinicalevaluation such as the determination of activation markers orimmunological analyses (e.g., assay for intracellular or extracellularinterleukins IL-1β, IL-2, IL-4, IL-6, IL-10 and IL-12, γIFN and TNFα).

Example 14

Liposome formulation. Liposomes suitable for parenteral administrationare prepared as follows. 400 mg of phosphatidyl choline and 80 mg ofBrEA are dissolved in chloroform and methanol (2:1 v/v) and the solutionis dried by rotary evaporation under reduced pressure. The resultingfilm is rehydrated by adding 8.0 mL of a 0.9% w/v NaCl solution andagitating the solution. The sizes of the liposomes are optionallymeasured, e.g., by photon correlation spectroscopy (Malvern Zetasizer3000 or equivalent). The liposomes are optionally sized by, e.g.,sonication to reduce the average size below 400 nm, or by filtrationusing suitable filters. Similar procedures are used to prepare liposomepreparations that contain a formula 1 compound at about 15-100 mg/mL.The formulation is used to deliver the compound orally or parenterally(I.M., S.C., I.V.).

Example 15

Cyclodextrin formulation. A cyclodextrin formulation containing BrEA isprepared as follows. 45 g of hydroxypropyl-β-cyclodextrin is added to 1L of sterile physiological saline and the mixture is stirred for about4-24 hours, until a clear solution is obtained. Non-micronized BrEA isadded to give a concentration of 20 mg/mL and the mixture is stirreduntil a clear solution is obtained. The solution is sterilized byfiltration using a 0.2 μm pore size filter and dispensed into sterilecontainers. Similar procedures are used to prepare cyclodextrinformulations that contain a formula 1 compound at about 15-100 mg/mL.The formulation is used to deliver the compound orally, parenterally(I.M., S.C., I.V.) or by a buccal or sublingual route.

Example 16

Suppository formulation. A suppository formulation containing a formula1 compound such as BrEA is prepared as follows. Sufficientnon-micronized BrEA is measured to obtain a desired number of units thatcomprise 500 mg each of BrEA. The BrEA is blended with with asuppository base, e.g., triglyceride from edible vegetable oil, toprovide desired characteristics, e.g., a free fatty acid content ofabout 0.1% w/w, a saponification value of about 242, an iodine value ofabout 3, moisture at about 0.1% w/w and a closed capillary melting pointof about 35° C.

Example 17

Human HCV clinical trial. A female patient infected with HIV and HCV wasdosed I.V. with BrEA for 3 consecutive days using a formulation thatcontained 20 mg/mL BrEA in 45% w/v hydroxypropyl-β-cyclodextrin andsaline. Four mL of the formulation (80 mg BrEA) was administered to thepatient every 4 hours during the 3-day treatment period. The patient'spredosing HCV level was 6.5 Log₁₀ as measured by PCR and the HCV levelwas 6.2 Log₁₀ on the first day of dosing, 5.5 Log₁₀ on the 3^(rd) day ofdosing and 4.9 Log₁₀ three days after the last dose was administered.HIV RNA levels as measured by PCR was 5.2 Log₁₀ (predosing), 5.8 Log₁₀(first day), 5.9 Log₁₀ (third day) and 5.4 Log₁₀ (day 6). The NK cellcounts (cells/mm³) were 28, 41 and 38 at predosing, day 0 and day 3.

Example 18

Formulation. A formulation comprising 100 mg/mL BrEA, ˜30% v/v PEG300,30% v/v propylene glycol, 30% v/v benzyl benzoate and 2% v/v benzylalcohol was prepared by suspending BrEA in polyethylene glycol 300, andsequentially adding propylene glycol and benzyl benzoate, to form asolution, which was diluted to the final desired volume with additionalpropylene glycol. The procedure is described below.

The calculated amount of polyethylene glycol 300 was added to acompounding vessel. Then, while mixing, the calculated amount of BrEAwas added to the vessel, and mixed for at least 5 minutes to form asmooth, creamy liquid propylene glycol was added to the vessel, andmixed for a minimum of 5 minutes to form a uniform suspension. Thecalculated amount of benzyl benzoate is added to the vessel, and mixedfor approximately 5 minutes to form a translucent liquid suspension.Propylene glycol was then added to achieve the desired finalformulation, and mixed for approximately 5 minutes. The drug solutionwas transferred to a volume dispensing device set to deliver 1.2 mL pervial. Under nitrogen pressure, the solution was filtered through two 0.2μm polyvinylidene fluoride filters in series into 2 cc amber glassvials. The vials were capped with Teflon-coated, butyl-rubber stoppersand crimp sealed.

Example 19

Opportunistic infection clinical protocol. A double blind, randomized,placebo controlled study of 100 mg of BrEA administered intramuscularlyto late stage HIV-infected patients at risk for opportunistic infections(OIs). HIV-1 seropositive patients with a CD4 cell count≦100 cells/mm³,HIV RNA at 1×10⁶ copies/mL and a Karnofsky score of at least 60 areidentified for potential inclusion into the protocol. Patients in allclinical protocols must understand and sign a written informed consentform prior to screening evaluations.

BrEA in the formulation of example 16 is used. Administration of drug orvehicle will be for 3 to 5 consecutive days followed by about 35-90daysof observation, e.g., 37 days of observation. An exemplary treatmentregimen comprises 5 days of treatment followed by 37 days ofobservation, which is repeated for a total of 7 courses over 42 weeks.The incidence rate of OIs as well as the time to resolution or controlof the OIs will be monitored and compared to a placebo control group.The patients may be monitored monthly for 2 or 3 months after completionof the study for follow-up. The incidence of OIs or conditionsassociated with AIDS are monitored, e.g., as tuberculosis (TB),candadiasis, Pneumocystis pneumonia (PCP), diarrhea, or Kaposi'ssarcoma, may be evaluated as protocol endpoints. If a patient isdiagnosed with one or more of the protocol specified opportunisticinfections, the protocol regimen a treatment for the OI will beinitiated, e.g., Fluconazole for Candidiasis or for PCP, trimethoprimand sulfamethoxazole or Dapsone. A similar protocol is used with otherformula 1 compounds.

Example 20

Human HIV clinical protocol. Patients infected with HIV are dosed withan i.m. injection of 25-200 mg of BrEA using a formulation containing100 mg/mL BrEA, PEG300 ˜30% v/v, propylene glycol 30% v/v, benzylbenzoate 30% v/v and benzyl alcohol 2% v/v. The patients are dosed onceper day for 5 consecutive days followed by a period of about 28 days orlonger with no BrEA treatment. The patients were them provided with onemore course of 5 consecutive days of dosing with BrEA, followed by anon-dosing period of at least about 28 days. Up to 5 rounds of 5-daytreatments, followed by at least 28 days of no dosing were provided.Immunological responses were then assayed using blood or plasma samplesfrom the patients by flow cytometry and other known analytical methods.Immune cell subsets or other measured markers were assayed within 24hours of obtaining the sample from each patient. Labeled antibodies,e.g., anti-CD antigen antibodies conjugated with fluorescent dyes (FITC,phycoerythrin, allophycocyanin or PerCP), were prepared and usedessentially according to standard protocols using commercially availablereagents, see, e.g., PharMingen, 1998 Research Products Catalog,technical protocols at pages 732-774, human cell surface molecules atpages 182-295 and mouse, rat and hamster cell surface molecules at pages2-173 and cytokine and chemokine reagents at pages 344-489.

The clinical protocol is a phase I/II, open-label, randomized study of 3dose levels of BrEA administered intramuscularly to HIV-infectedpatients who are treatment naive. There will be 3 treatment groups andeach group will consist of 2 parts (Parts A and B). Patients willreceive the same dosage of BrEA throughout Parts A and B of the study.If a patient experiences an antiviral response (an HIV RNA titer atleast 0.5 log below the average of the screening and baseline values) orbenefits (any decrease in HIV RNA titers below the average of thescreening and baseline values) from the treatment received during PartsA and B of the study, the patient may continue receiving 5-day treatmentcourses of the BrEA formulation of example 2 at the dose initiallyreceived. This treatment course may be repeated up to six times.

All patients may be monitored for levels of HIV RNA (Chiron Quantiplex™branched chain DNA assay), T-cell subsets [CD4/CD8], proviral HIV DNA(PBMC), interleukins [IL-2, 4, 6, 8, 10, and 12] (serum), γIFN (serum),insulin-like growth factor [IGF-1] (serum) and tumor necrosis factor[TNF] (serum) throughout the study. PBMC quantitative co-culture (cells)may be conducted on a subset of patient samples. Assays for additionalactivation markers may be conducted. Analysis of chemistry andhematology panels and urinalysis is planned. Additionally, patientsco-infected with hepatitis B and/or C viruses, malaria or tuberculosismay be monitored regularly for viral titers or microbiological cultures.Serial blood and urine samples will be collected from a subset ofpatients for pharmacokinetic determination after the first dose on PartA and the last dose on Part B.

Treatment may consist of more than one intramuscular injection.Intramuscular injections may be administered in different locations(i.e., left or right upper arms or thighs or buttocks) and a single 100mg or 200 mg dose of BrEA may be delivered to patients in two or moresubdoses of less than 100 mg (e.g., 50 mg).

There are two segments of this study, Segment 1 and 2. Both segmentsconsist of two parts, Part A and Part B. The first 12 patients enrolledon the study will be assigned to the design described in Segment 1. Theremaining 24 patients will be assigned to Segment 2 of the study. Thedesign of each segment is provided below.

Part A will consist of a single intramuscular injection of a BrEAformulation. The day the patient receives the injection will be studyday 1. Patients participating in the pharmacokinetic subgroup will haveserial blood and urine samples collected, beginning on study day 1. PartB of the study begins on study day 8 (Segment 1) or study day 15(Segment 2).

Segment 1 Part B consists of 5 consecutive daily intramuscularinjections of the formulation of example 1 at the same dose as receivedin Part A of the study. The day the patient receives the first dose willbe on about study day 8-12. The 5-day treatment course is followed by anapproximate 28-day observation period (or approximately 32 days from afirst dose on day 8 to the initiation of a second treatment course onday 40). During the observation period, patients will be asked to returnto the clinic on a weekly basis for various tests. Patientsparticipating in the pharmacokinetic subgroup will have serial blood andurine samples collected, beginning approximately on study day 12-17.

Segment 2 Part B consists of 5 consecutive daily intramuscularinjections of the formulation of example 2 at the same dose the patientreceived during Part A of the study. The day the patient receives thefirst dose will be about at study day 15. The 5-day treatment course isfollowed by an approximate 45 day observation period (or approximately49 days from the first dose on study day 15 to the initiation of thenext treatment course on study day 64). During the observation period,patients will be asked to return to the clinic on a weekly basis forvarious tests. Patients participating in the pharmacokinetic subgroupwill have serial blood and urine samples collected, beginningapproximately on study day 19.

Randomization in this dose escalation study is as follows. When 4 of the12 patients per treatment group have completed 5 days of daily dosing onPart B and have not experienced a serious drug-related adverse event,enrollment into the next higher dose level will occur, afterconsultation between the sponsor and investigators.

The first four patients enrolled will be assigned to the 50 mg dosegroup. If no serious drug-related adverse events are experienced, thenext 8 subjects will be randomized to either the 50 mg or 100 mg doselevel in a 1:1 fashion. If no serious drug-related adverse events occurin patients receiving 100 mg, then the next 24 patients will berandomized to either the 50,100, or 200 mg dose group in a 1:2:3fashion.

If 4 of the 12 patients in a dose group experiences a seriousdrug-related event (Grade III or IV), 2 additional patients will beenrolled at the same dose level. Additionally, patient enrollment on tothe next dose level, if enrolling, will be temporarily on hold untilsafety is assessed. If one of the 2 additional patients experiences aserious drug-related event, dosing in this dose level will discontinue.Upon consultation with the sponsor and investigators, additionalpatients may be enrolled at a dose between the dose-limiting group andthe next lower dose group to determine the maximum tolerated dose (MTD).Enrollment of additional patients at a specific dose level will bedetermined in a protocol amendment.

The results indicated that a single 50 mg or 100 mg dose of BrEAincreased the numbers of activated CD8⁺ and CD4⁺ T cells (e.g., CD8⁺,CD69⁺, CD25⁻ cells) that were circulating in the patient's blood. Also,the circulating numbers of dendritic precursor cells, NK cells, LAKcells and cells that mediate ADCC (antibody-dependent cell-mediatedcytotoxicity mediated by the CD8⁺, CD16⁻ immune cell subset) functionswere increased. Further increases were usually observed on dosing for 5consecutive days.

Some of the results are summarized below. Course 1, 2 and 3 refer toeach 5 consecutive day treatment regimen of one daily injection withBrEA (50 or 100 mg BrEA per injection). In the diagrams below, HE2000refers to the formulation containing 100 mg/mL BrEA, PEG300 ˜30% v/v,propylene glycol 30% v/v, benzyl benzoate 30% v/v and benzyl alcohol 2%v/v. The data shown below was obtained from patient blood samples atbaseline (on the day dosing was initiated) and at various times afterthe patients received at least one dose of BrEA. The results showedsignificant increases in immune cell populations and cytokine expressionprofiles associated with Th1 responses. The patients in this protocolinitially had CD4 counts of at least 200 per mm³ and a serum HIV RNAload of 5,000 to 1×10⁶ RNA copies/mL. After dosing with one course ofBrEA (5 consecutive daily i.m. injections), all patients showedincreases in levels of immune cells including activated CD8 T cells(e.g., CD8⁺, CD69⁺, CD25⁻), LAK cells (e.g., CD8⁺, CD16⁺, CD38⁺), NKcells (e.g., CD8⁻, CD16⁺), ADCC cells (e.g., CD8⁻, CD16⁺) and dendriticcells (Lin⁻, HLA-DR⁺, CD11c⁺ or Lin⁻, HLA-DR⁺, CD123⁺). Average CD4IL-10 production dropped from a median of 66% to 4% of the cells, whileCD4 IFNγ went from a median of 8% to 63%, leading to a Th2 to a Th1shift in cytokine production.

In the tables shown below baseline data is indicated by “BL” or by“pre”.

Increased immunophenotypes after BrEA therapy Phenotype Baseline^(a)Course 1 Course 2 Course 3 CD8+CD69+CD25− 18 54 56 75 n = (13) (13) (9)(4) ^(b)p = <0.001 <0.001 0.04 CD8+CD16+CD38+ 8 27 28 25 n = (10) (10)(4) (4) p = <0.001 0.047 0.02 CD8−CD16+ 53 253 288 249 n = (12) (12) (4)(2) p = <0.001 0.02 0.04 Lin-HLA-DR+ 3.2 17.7 11.4^(c) 14.7^(c)CD11c+/CD123+ n = (10) (10) (5) (4) p = <0.001 0.02 0.04 IL2+CD4^(d)3.14^(e) 29.25 31.42 13.59 n = 13 13 3 4 p = <0.001 0.09 0.04IL10+CD4^(d) 66 20.9 8.9 15.3 n = 13 13 5 3 p = 0.005 0.005 0.03 Th1Response^(d) 17 66 64 53 n = (13) (13) (5) (5) p = 0.001 0.033 0.025^(a)Median values of cells/μL ^(b)paired value t test ^(c)Test notavailable at baseline for patients receiving second and third courses,baseline value from initiation of 2^(nd) course = 6.4 ^(d)% of CD4^(e)Baseline values from day 8 (preceding the first five-day treatment)

Median activated T cells (CD8⁺CD69⁺CD25⁻) vs. baseline by course Course1 baseline 19 cells/μL Course 1 53 cells/μL n = 13, p < 0.001 Course 2baseline 19 cells/μL Course 2 54 cells/μL n = 9, p < 0.001 Course 3baseline 19 cells/μL Course 3 74 cells/μL n = 4, p = 0.04 Median LAKresponse (CD8⁺CD16⁺CD38⁺) vs. baseline, responders, by course Course 1baseline 8 cells/μL Course 1 26 cells/μL n = 10, p < 0.001 Course 2baseline 12 cells/μL Course 2 27 cells/μL n = 4, p = 0.04 Course 3baseline 12 cells/μL Course 3 25 cells/μL n = 4, p = 0.02 Median NK,ADCC (CD8⁻CD16⁺) responders vs. baseline by course Course 1 baseline 52cells/μL Course 1 255 cells/μL n = 12, p < 0.001 Course 2 baseline 59cells/μL Course 2 291 cells/μL n = 4, p = 0.02 Course 3 baseline 56cells/μL Course 3 249 cells/μL n = 2, p = 0.04 Median dendritic cellresponse (Lin⁻HLA-DR+CD123⁺/CD11c⁺) by course Course 1 baseline 3.2cells/μL Course 1 17.7 cells/μL n = 10, p = 0.001 Course 2 baseline 6.6cells/μL Course 2 11.6 cells/μL n = 5, p = 0.02 Course 3 baseline 6.3cells/μL Course 3 14.7 cells/μL n = 4, p = 0.04 16α-Bromoepiandrosteronenormalizes IL-10⁺cells in HIV-infected patients % of CD4⁺ cells that areIL10⁺ Normals (HIV⁻) 8% n = 6 HIV⁺ patients 64% n = 13, p < 0.001treated HIV⁺ patients 4% n = 13, p < 0.001 16α-Bromoepiandrosteroneincreases Th1 cell proportion in HIV-infected patients median % of CD4⁺cells that are IFNγ⁺and IL10⁻ Normals (HIV⁻) 43% HIV⁺ patients 8% p <0.05 treated HIV⁺ patients 63% p < 0.001 Median Th1 (IFNγ⁺ dominance)response vs. baseline median % of IFNγ⁺ T cells Course 1 baseline 17%Course 1 66% n = 13, p = 0.00 Course 2 baseline 5% Course 2 64% n = 4, p= 0.0 Course 3 baseline 5% Course 3 53% n = 4, p = 0.0

Example 21

Treatment of symptoms of HIV infection. Two HIV patients with chronicdiarrhea were dosed with BrEA as follows. A BrEA formulation (40 mg/mLBrEA in 25% v/v PEG 300,12.5% v/v ethanol, 5% v/v benzyl benzoate,˜57.5% v/v propylene glycol) was delivered subcutaneously. The patientsreceived 60 mg of BrEA in 1.5 mL daily for 10 days. During the period ofdosing, the diarrhea ceased. After the 10-day dosing period ended,diarrhea resumed. In other patients receiving oral BrEA, diarrhea alsowent into remission.

Example 22

Subcutaneous formulation. A BrEA formulation was prepared essentially asdescribed herein. The formulation contained 50 mg/mL BrEA, 40% v/v PEG200, 2% v/v benzyl alcohol, 2% v/v benzyl benzoate and ˜66% v/vpropylene glycol (qs). The formulation is particularly suitable forsubcutaneous administration of the compound.

Example 23

Preparation of BrEA hemihydrate—procedure 1. Crude BrEA was prepared bybromination of epiandrosterone, followed by crystallization frommethanol. The hemihydrate was prepared by dissolving 25 g of crude BrEAin 75 mL of refluxing ethanol with moderate agitation. To the BrEAsolution 12.5 mL of water was slowly added while maintaining thesolution at reflux with agitation. Agitation of the solution wasmaintained and the solution was then allowed to cool to about 20-25° C.and kept at about 20-25° C. for about 15 minutes to obtain a suspensionof BrEA hemihydrate crystals. The crystals were recovered by filtration,washed with a solution of 25 mL of water:ethanol (5:1 v/v) at about20-25° C. and then vacuum dried for about 13 hours at 50-60° C. untilthe product weight was constant. The crystals were primarily rod andneedle shaped, with smaller amounts of other shapes such as tablets.

The procedure gave 22.5 g of BrEA hemihydrate (yield 90%) with a watercontent of 2.6% w/w by KF analysis, a purity of 100% by HPLC areaanalysis, an FTIR spectrum with carbonyl peaks at 1741 cm⁻¹ and 1752cm⁻¹. The FTIR scan of anhydrous BrEA shows a single carbonyl peak at1749 cm⁻¹. The DSC scan showed three endotherms. One had a broad shallowpeak with an onset at about 109-110° C. and ending at about 150° C. Thisbroad DSC peak is consistent with the loss of water from the hemihydratecrystals as the temperature of the sample increased. The secondendotherm at about 83-100° C. is consistent with the loss of the smallamount of residual ethanol from the sample. A DSC scan of anhydrous BrEAdoes not have the broad endotherm that is observed with the hemihydrate.Also consistent with the loss of water from the hemihydrate over the100-150° C. range is a sharp third endotherm peak in the hemihydrate DSCscan at about 163-164° C., which is the melting point of anhydrous BrEA.The FTIR was obtained using USP method <197>, where the BrEA hemihydratesample was prepared in KBr. The DSC thermogram was obtained by scanningfrom 25° C. to 250° C. with a heating rate of 10° C./minute.

Example 24

Preparation of BrEA hemihydrate—procedure 2. The hemihydrate wasprepared by dissolving 10 g of crude BrEA in 40 mL of refluxing acetonewith moderate agitation. To the BrEA solution 4.0 mL of water was slowlyadded while maintaining the solution at reflux with agitation. Agitationof the solution was maintained and the solution was then allowed to coolto about 20-25° C. and kept at about 20-25° C. for about 15 minutes toobtain a suspension of BrEA hemihydrate crystals. The crystals wererecovered by filtration, washed with a solution of 6.0 mL ofwater:acetone (10:1 v/v) at about 20-25° C. and then vacuum driedovernight (about 13-15 hours) at 50-60° C. until the product weight wasconstant. The procedure gave 7.0 g of BrEA hemihydrate (yield 70%) witha water content of 2.6% w/w by KF analysis and an FTIR spectrum withcarbonyl peaks at 1741 cm⁻¹ and 1752 cm⁻¹.

Example 25

Analysis of BrEA hemihydrate particle size. BrEA hemihydrate crystalswere prepared essentially as described herein and sized using a particlesizing apparatus (Malvern Instruments). The analysis model used was fora polydisperse sample and a volume distribution type. The analysisshowed a range of crystal diameter sizes from about 0.5 μm to about 880μm. About 90% of the crystals had a diameter of about 20 μm to about 220μm and the majority of the crystals had a diameter of about 30-200 μm.The mean crystal diameter was about 93 μm. The specific surface area ofthe crystals was about 0.25 m²/g.

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any of the various specific embodiments,compounds or compositions described herein may be further modified toincorporate other appropriate features, e.g., as shown in any other ofthe specific embodiments disclosed herein or in the cited references.

1. A pharmaceutical formulation comprising one or more excipients and acompound having the structure

wherein the hydrogen atom at the C5-position, if present, is in the α-or β-configuration; R¹ is —OH; R², R³ and R⁴ independently are —OH or anester; R⁵ is —CH₃ or —CH₂OH; and R⁶ is —H, —CH₃ or —CH₂OH.
 2. Thepharmaceutical formulation of claim 1 having the structure


3. The pharmaceutical formulation of claim 2 wherein R¹, R², R³ and R⁴are —OH.
 4. The pharmaceutical formulation of claim 1 wherein R⁵ and R⁶are —CH₃.
 5. The pharmaceutical formulation of claim 3 wherein R⁶ is —Hand R⁵ is —CH₃ or —CH₂OH.
 6. The pharmaceutical formulation of claim 2wherein R² and R⁴ are —OH and R³ is —OH, —O—C(O)—CH₃, —O—C(O)—C₂H₅ or—O—C(O)—CH₂CH₂CH₃.
 7. The pharmaceutical formulation of claim 1 havingthe structure


8. The pharmaceutical formulation of claim 7 wherein R¹, R², R³ and R⁴are —OH and the pharmaceutical formulation is an oral formulation. 9.The pharmaceutical formulation of claim 7 wherein R⁵ is —CH₂OH.
 10. Thepharmaceutical formulation of claim 7 wherein R⁶ is —H and R⁵ is —CH₃ or—CH₂OH.
 11. The pharmaceutical formulation of claim 7 wherein R² and R⁴are —OH and R³ is —OH, —O—C(O)—CH₃, —O—C(O)—C₂H₅ or —O—C(O)—CH₂CH₂CH₃.12. The pharmaceutical formulation of claim 11 wherein R³ is—O—C(O)—CH₃, —O—C(O)—C₂H₅ or —O—C(O)—CH₂CH₂CH₃.